THE  PROPERTY  OF 

H9lii!8Miiiil'"  -'■"  icftliePaGil, 


MEPICAL    .SCInI®OL 


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Hateo'inK-'-'^'-llglfionk  Pacific 


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ATLAS  AND  ESSENTIALS 


OF 


BACTERIOLOGY 


BY 

Prof.  K.  B.  LEHMANN 

CHIEF  OP  THE   HYGIENIC  INSTITUTE   IN   WUrZBURG 
AND 

Dr.  EUDOLF  NEUMANN 

ASSISTANT  IN  THE  HYGIENIC  INSTITUTE  IN  wtJRZBURG 


WITH  63  CHROMO-LITHOGRAPHIC  PLATES,  COMPRISING 
558  FIGURES,  AND  NUMEROUS  ENGRAVINGS 


NEW  YORK 
WILLIAM  WOOD   AND  COMPANY 


•   •        •    • 


.97 


LIST   OF   PLATES. 


Plate  1.-— Micrococcus  pyogenes  a  aureus.      (Ros.)      Lehm 
and  Neura. 
(Staphylococcus  pyogenes  aureus.     Rosenbach.) 
Plate  2.  —Micrococcus  pyogenes  y  albus.      (Ros. ) 

(Staphylococcus  pyogenes  albus.     Rosenbach.) 

Micrococcus  pyogenes  /3  citreus.     (Ros.) 
(Stai:^ylococcus  pyogenes  citreus.     Rosenbach. ) 

Micrococcus  candicans.     Flilgge. 
Plate  3. —Micrococcus  agilis.     Ali  Cohen. 

Micrococcus  gonorrhoeae.     Neisser,  Bumm. 

Streptococcus     meningitidis    cerebrospinalis.       (Weichs. ) 
Lehm.  and  Neum. 
Plate  4. — Micrococcus  roseus.     (Bumm.)     Lehm.  and  Neum. 
Plate  5.— Streptococcus  lanceolatus.     Gamaleia. 

(Diplococcus  pneumoniae.     A.  Fraenkel.) 
Plate  6.  — Streptococcus  pyogenes.     Rosenbach. 
Plate  7. — Micrococcus  tetragenus.     Koch,  Gaffky. 
Plate  8.— Micrococcus  luteus.     Cohn  em.  Lehm.  and  Neum. 

Sarcina  pulmonum.     Virchow,  Hauser. 
Plate  9. — Sarcina  flava.     De    Bary  em.  Lehm.  and    Stuben- 

rath. 
Plate  10. —Sarcina  aurantiaca.     Flligge. 
Plate  11. — Sarcina  cervina.     Stubenrath. 

Sarcina  pulmonum.     Virchow. 

Sarcina  erythromyxa.     Krai. 

Sarcina  lutea.     Fliigge. 

Sarcina  aurantiaca.     Flugge. 

Sarcina  rosea.     Schroeter  em.  Zimmermann. 

Micrococcus  badius.     Lehm.  and  Neum. 

Sarcina  canescens.     Stubenrath. 


13566 


IV 


LIST   OF    PLATES. 


Plate  12.  —Bacterium  pneumonias.     Friedlander. 
Plate  13. — Bacterium  acidi  lactici.     Hiippe. 

(Lactic  acid  bacillus. ) 
Plate  14.— Bacterium  coli  commune.     Escherich. 
Plate  15. — Bacterium  coli  commune.     Escherich. 
Plate  16.— Bacterium  typhi.     Eberth,  Gaffky. 

(Typhoid  bacillus.) 
Plate  17. — Bacterium  typhi.     Eberth,  Gaffky. 
Plate  18. — Bacterium  septicsemise  hsemorrhagicae.     Hiippe. 

(Chicken  cholera,  rabbit  septicaemia,  etc. ) 
Plate  19. — Bacterium  mallei.     Loffler. 

(Glanders  bacillus.) 
Plate  20. — Corynebacterium  diphtherise.      (Loffler.)      Lehm. 
and  Neum. 
(Diphtheria  bacillus.) 
Plate  21. — Bacterium  latericium.     Adametz. 

Bacterium  hsemorrhagicum.      (Kolb.)     Lehm.  and  Neum. 
(Morbus  Werlhofii. ) 
Plate    22.      Bacterium      putidum.       (Flugge.)      Lehm.     and 

Neum. 
Plate  23.— Bacterium    syncyaneum.      (Ehrenb.)      Lehm.    and 
Neum. 
(Bacillus  cyanogenes  Flugge.     Blue  milk. ) 
Plate  24. — Bacterium  syncyaneum.      (Ehrenb.)      Lehm.    and 
Neum. 
(Bacillus  cyanogenes  Flugge.     Blue  milk.) 
Plate  25. —Bacterium  prodigiosum.      (Ehrenb.)     Lehm.  and 

Neum. 
Plate  26. —Bacterium    kiliense.         (Breunig     and     Fischer.) 
Lehm.  and  Neum. 
(Kiel  water  bacillus. ) 
Plate  27. — Bacterium  janthinum.     Zopf. 

Plate  28. — Bacterium    fluorescens.       (Flugge.)       Lehm.    and 
Neum. 
(Bacillus  fluorescens  liquefacieus  Flugge, ) 
Plate  29. — Bacterium  pyocyaneum.       (Flilgge. )      Lehm.  and 
Neum. 
(Green  pus.) 
Plate  30. — Bacterium  Zopfii.     Kurth. 


LIST    OF    PLATES.  V 

Plate  31. — Bacterium  Zopfii.     Kurth. 

Plate  32. —Bacterium  vulgare  ^  mirabilis.     (Hauser. )     Lehm. 
and  Neum. 
(Proteus  mirabilis  Hauser.) 
Plate    33.  —  Bacterium     vulgare.       (Hauser.)      Lehm.     and 
Neum. 
(Proteus  vulgaris  Hauser. ) 
Plate    34. — Bacterium     erysipelatus    suum.      (Loffler.)      Mi- 
gula. 
(Hog  erysipelas. ) 

Bacterium  murisepticum.      (Flligge.)     Migula, 

(Mouse  septicaemia. ) 
Plate  35. — Bacillus  megatherium.     De  Bary. 
Plate  36.— Bacillus  subtilis.     F.  Cohn. 

(Hay  bacillus, ) 
Plate  37.— Bacillus  subtilis.     F.  Cohn. 

(Hay  bacillus.) 
Plate  38. — Bacillus  anthracis.     F.  Cohn  and  R.  Koch, 

(Anthrax  bacillus. ) 
Plate  39. — Bacillus  anthracis.     F.  Cohn  and  R.  Koch. 

(Anthrax  bacillus. ) 
Plate  40. — Bacillus  anthracis.     F.  Cohn  and  R.  Koch. 

(Anthrax  bacillus. ) 
Plate  41.  — Bacillus  mycoides.     Fliigge. 

(Root  bacillus.) 
Plate  42.  Bacillus  mycoides.     Flugge. 

(Root  bacillus.) 

Bacillus  butyricus.     Hlippe. 

(Butyric  acid  bacillus.) 
Plate  43.— Bacillus  vulgatus.     (Flugge.)     Migula. 

(B.  mesentericus  vulgatus  Flugge.     Potato  bacillus. ) 
Plate  44.— Bacillus   mesentericus.       (Flugge.)       Lehm.    and 
Neum. 

(B.  mesentericus  fuscus  Fliigge.) 
Plate  45.— Bacillus  tetani.     Nicolaier. 

(Tetanus  bacillus.) 
Plate  46.— Bacillus  Chauvoei  of  French  writers. 

(Rauschbrand.) 
Plate  47.— Bacillus  oedematis  maligni.     Koch. 


VI 


LIST   OF    PLATES. 


iberculosi 

is.     (Koch.) 

Lehm,  and 

(Koch. ) 

Buchner, 

(Koch. ) 

Buchner. 

(Koch.) 

Buchner. 

(Koch.) 

Buchner. 

(Koch.) 

Buchner. 

. 

Plate  48. — Mycobacterium 
Neum. 
(Tubercle  bacillus. ) 
Plate  49.  — Vibrio  cholera). 

(Comma  bacillus. ) 
Plate  50. — Vibrio  cholera3. 

(Comma  bacillus. ) 
Plate  51. — Vibrio  choleras. 

(Comma  bacillus.) 
Plate  52. — Vibrio  cholerse. 

(Comma  bacillus.) 
Plate  53. — Vibrio  choleraB. 

(Comma  bacillus.) 

Vibrio  Metschnikovii.     Gamaleia. 

Vibrio  proteus.     Buchner. 

(Vibrio  Finkler.     Author. ) 
Plate  54. — Vibrio  albensis.     Lehm.  and  Neum. 

(Fluorescent  Elbe  vibrio. ) 
Plate  55. — Vibrio  danubicus  Heider. 

Vibrio  berolinensis  Rubner. 

Vibrio  aquatilis  Gunther. 
Plate  56. — Vibrio  proteus.     Buchner. 

(Vibrio  Finkler.     Author.) 
Plate  57. — Spirillum  rubrum.     v.  Esmarch. 

Spirillum  concentricum.     Kitasato. 
Plate  58. — Spirillum  serpens.      (E.  O.  Mijller. ) 
Neum. 

Spirilla  from  nasal  mucus. 

Spirillum  undula.     Ehrenberg. 

Vibrio  spermatozoides.     Loffler. 

Spirochsetes  of  the  mucus  from  the  gums. 

Spirillum  Obermeieri  Virchow. 

(Recurrens  spirilla. ) 
Plate  59.— Leptothrix  epidermidis.     Biz. 
Plate  60.— Oospora  farcinica.     Sauv.  and  Rad. 

(Farcin  de  boeuf . ) 
Plate  61. — Oospora  chromogenes.      (Gasparini.) 
Neum. 

(Cladothrix  dichotoma  Autorum  non  Cohn. ) 


Lehm.  and 


Lehm.  and 


LIST    OF    PLATES.  711 

Plate  62.— Oospora  bovis.     (Harz. )     Sauv.  and  Rad. 

(Actinomyces. ) 
Plate  63. — Mycobacterium   leprae.      (Arm.   Hansen.)      Lehm. 
and  Neum. 

(Leprosy  bacillus.) 

Bacterium  influenzae.     R.  Pfeiffer. 

(Influenza  bacillus. ) 

Bacterium  pestis  (Kitasato,  Yersin) .     Lehm.  and  Neum. 

(Plague  bacillus. ) 

Bacteria  in  soft  chancre. 


LIST   OF  ABBEEVIATIONS. 


A.  H.  =  Archiv  flir  Hygiene,  Munich.     Oldenbourg  since  1883. 

A.  G.  A.  =  Arbeiten  aus  dem  kaiserlichen  Gesundheitsamt, 
Berlin,  Springer,  since  1885. 

A.  K.  =  Arbeiten  aus  dem  bakteriologischen  Institut  der  tech- 
nishen  Ilochschule  zu  Karlsruhe.  Edited  by  Klein  and 
Migula,  since  1894. 

A.  P.  —  Annalcs  de  1' Institut  Pasteur,  Paris,  Masson,  since  1887. 

C.  B.  =  CentralblattfiirBakteriologieuudParasitenkunde,  Jena, 
Fischer.  Since  1894  this  publication  has  been  divided  into 
two  parts : 

C.  B.,  Part  I.,  devoted  to  medico-hygienic  questions. 

C.  B.,  Part  II.,  devoted  to  zymotechnical,  agricultural,  and 
phytopathological  studies. 

Z.  H.  =  Zeitschrift  fiir  Hygiene,  Leipsic,  Veit,  since  1886. 

Fltigge  =  Fliigge  :  Die  Mikroorganismen,  second  edition,  Leip- 
sic, 1886. 

Kitt,  B,  K.  =  Kitt :  Bakterienkunde  fiir  Tieraerzte,  second  edi- 
tion, Vienna,  1893. 

Zimmermann  1  and  2  =  0.  E.  R.  Zimmermann  :  Die  Bakterien 
unserer  Trink-  und  Nutzwasser,  Chemnitz,  Part  I.,  1890; 
Part  II..  1894. 


Tab.     1, 


Explanation  of  Plate  1. 

Micrococcus   pyogenes  a  aureus.      Eosenbach,  Leh- 

mann  and  Neumann. 

(Staphylococcus  aureus  Eos.) 

I.  Gelatin  stick  culture,  six  days  at  22°. 
II.  Agar  streak  culture,  five  days  at  22°. 

III.  Agar  stick  culture,  five  days  at  22°.     Stick  canal. 

IV.  Agar  stick  culture,  five  days  at  22°.     Surface. 
V.  Agar  plate  culture  (natural  size),  six  days  at  22°. 

Superficial  and  deep  colonies. 

VI.  Agar  plate,  six  days  at  22"".      x60.     Superficial 
small  colony. 

VII.   Gelatin  plate  (natural   size),  four   days   at   22°. 
Superficial  and  deep  colonies. 
VIII.  Gelatin  plate,  four  days  at  22"".     x  60.    Superficial 
and  deep  colonies. 

IX.  Potato  culture,  six  days  at  22°. 
X.  Microscopical  preparation  ( X  1, 000)  of  agar  cul- 
ture, two  days  at  22°. 

XI.  Microscopical  preparation;    individual  cocci,  be- 
fore and  after  division.      xlj500. 

XX. 


Explanation  of  Plate  2. 

Micrococcus  pyogenes  y  albus.     Eosenbach. 

(Staphylococcus  albus.) 

I.  Agar  streak  culture,  four  days  at  22°. 
II.  Gelatin  stick  culture,  fi-ve  days  at  22°. 

Micrococcus  pyogenes  /5  citreus.     Eosenbach. 
(Staphylococcus  citreus.) 

III.  Agar  streak  culture,  six  days  at  22°. 

Micrococcus  candicans.  Flligge. 

IV.  Gelatin  stick  culture,  six  days  at  22°. 
V.  Gelatin  plate,  eight  days  at  22°. 

VI.  Gelatin  plate,  six  days  at  22°.     Left  side,  super- 
ficial colony ;  right  side,  deep  colony,      x  50. 
VII.  Potato  culture,  ten  days  at  22°. 
VIII.  Microscopical  preparation  of  agar  culture  (xTOO), 
two  days. 


Tab.     2. 


\^ 


LithAlSt  V  y  RpirMiold  Vliiiubcii 


Tab.     3. 


LiihAnsr.v.  K  Reirhhold .  Miinchm 


Explanation  of  Plate  3. 

Micrococcus  agilis.     Ali-Cohen. 

I.  Gelatin  stick  culture,  six  days  at  22°. 
II.  Gelatin  plate,  seven  days  at  22°.     x  50.    On  right 
side,   superficial   colony;    on   left   side,   deep- 
seated  colony. 

III.  Agar  plate,  seven  days  at  22°.     Natural  size. 

IV.  Microscopical  preparation  (x600)  from   an  agar 

culture  two  days  old.       The  individual  cocci 
vary  greatly  in  size,  and  are  more  irregular  than 
appears  in  the  plate. 
V.  Potato  culture,  ten  days  at  22°. 

Micrococcus  gonorrhce^.     Neisser,  Bumm. 

VI.  Smear  preparation  from  gonorrhoeal  pus.  x  1, 000. 
The  large  blue  cells  are  pus  cells. 

VI.  a.  Smear  preparation  from  gonorrhoeal  pus.  x 
1,200.     Semi-schematic. 

VI.  h.  Diplococcus  gonorrhoeae  much  enlarged.  Sche- 
matic. 

Streptococcus   meningitidis   cerebrospinalis. 
(Weichselbaum)  Lehmann  and  Neumann. 

VII.  Smear  preparation  from  meningeal  exudation ;  pus 
cells  with  transversely  divided  diplococci.  (Cop- 
ied from  Jaeger:  Zschr.  f.  Hyg.,  Vol.  XIX., 
PI.  VI.,  Pig.  3.)     About  X  1,200. 

VIII.  Microscopical  preparation;  pure  culture,  forma- 
tion of  tetrads.  Aboutx  1,200.  (Copied  from 
Jaeger:  Zschr.  f.  Hyg.,  Vol.  XIX.,  PI.  VII., 
Fig.  6.) 


VI.  a  Yl.  b 


Explanation  of  Plate  4. 

Micrococcus  roseus.    (Bumm)  Lehmann  and  Neumann. 

I.  Gelatin  stick  culture,  twenty  days  at  temperature 
of  room. 
II.  Agar  streak  culture,  thirty  days  at  temperature  of 
room.     The  white  reflex  on  the  right  side  is 
not  always  so  pronounced. 

III.  Agar  stick  culture,  ten  days  22°.     Puncture  canal. 

IV.  Agar  stick  culture,  ten  days  22°.     Surface. 

V.  Agar  plate,  twelve  days  at  22°.      x50.     Above,  a 

superficial,  below,  a  deep-seated  colony. 

VI.  Agar  plate.     More  delicate  structure.     Fourteen 

days  at  22°.    x  50.     Above,  a  superficial  colony, 

below,  deep-seated  colonies. 

VII.  Gelatin  plate,  eight  days  at  22°.     x  50.    Superficial 

and  deep  colonies. 
VIII.  Microscopical  preparation   from  agar  culture  (x 
1,000),  three  days.     The  cocci  are  dividing. 
IX.  Potato  culture  of  diplococcus  roseus  placed  on  an 
anthrax   culture,  ten   days  at  temperature   of 
room. 
X.  Potato  culture,  twenty  days  at  temperature  of 
room. 


Tab.     4. 


Tab.     5. 


Explanation  of  Plate  5. 

Streptococcus  lanceolatus.     Gamaleia. 

(Diplococcus  pneumoniae  A.  Fraenkel.) 

(Pneumococcus.) 

I.  Gelatin  stick  culture,  ten  days  at  22°. 
II.  Agar  streak  culture,  four  days  at  37°. 

III.  Agar  stick  culture,  four  days  at  37°.     Puncture 

canal. 

IV.  Agar  stick  culture,  four  days  at  37°.     Surface. 
V.  Agar  plate,  three  days  at  37°.     Natural  size. 

VI.  Agar  plate,  three  days  at  37°.      x50.     Superfi- 
cial colony.     The  dark  colony  is  situated  near 
the  surface. 
VII.  Agar  plate,  three  days  at  37°.     x50.    Deep-seated 
colonies. 
VIII.  Gelatin  plate,  eight  days  at  22°.     The  upper  col- 
ony superficial,  the  two  lower  ones  deep  seated. 
IX.   Smear    preparation    from    pneumonia    sputum. 

X  1,000. 
X.  Pure  culture  from  agar  plate  three  days  old.      x 
1,000. 
XI.  Microscopical  preparation. 

(a)  Diplococci,    single  and  arranged   in   chains. 
High  magnifying  power. 

(b)  Diplococci  surrounded  with   gelatinous   cap- 
sule. 

; .  ^ ! 


I  • 

XI. 


Explanation  of  Plate  6. 

Streptococcus  pyogenes.     Eosenbach. 

I.  Agar  streak  culture,  ten  days  at  37°. 
II.  Gelatin  stick  culture,  six  days  at  22°.     The  col- 
ony is  not  often  found  in  such  a  vigorous  state. 

III.  Agar   stick  culture,  six  days  at  37°.     Puncture 

canal. 

IV.  Agar  stick  culture,  six  days  at  37°.     Surface. 
V.  Gelatin  plate,  six  days  at  22°. 

VI.  Gelatin  plate,  six  days  at  22°.  x70.  Somewhat 
abnormal  shape  with  ragged  edges.  The  larger 
colonies  superficial,  the  smaller  ones  deep. 
VII.  Gelatin  plate,  six  days  at  22°.  x  70.  More  fre- 
quent form.  Upper  one  superficial,  lower  one 
deep. 
VIII.  Agar  plate,  eight  days  at  37°.  x50.  Larger  colony 
superficial,  smaller  colonies  deep. 

IX.  Microscopical    preparation    from  a  bouillon   cul- 
ture, two  days  at  37°.      X  700.     The  individual 
cocci  are  usually  more  regularly  rounded. 
X.  Microscopical  preparation  from  an  agar  culture, 
two  days.     Shorter  chains.      xljOOO. 

XI.  Microscopical  preparation.  Called  streptococcus 
conglomeratus.  Smear  preparation  from  the 
blood  of  the  spleen  from  a  case  of  scarlatina. 
Copied  from  Kurth  (Kaiserl.  Gesundheits- 
amt,  Vol.  VII.). 
XII.  Streptococci  chains,  before  and  during  division. 
High  magnifying  power. 

V 


V 


XII. 


Tab.     6. 


^^^^^^_ 

.,.: 

# 

e 

e 

i 
vn. 

•* 

1 

VI. 

LiiiuuiiL.v. r  itcicimuKi, Muartiea 


Tab.     7, 


LiilijViist  V  Y  RcirhhnUl.Miifirheti 


Explanation  of  Plate  7. 

Micrococcus  tetra genus.     Koch,  Gaffky. 

I.  Agar  streak  culture,  five  days  at  37°. 
II.  Gelatin  stick  culture,  ten  days  at  22°.  Puncture 
canal.     The  "  nail-head"  shape  is  characteristic. 

III.  Gelatin  stick  culture,  ten  days  at  22°.     Surface. 

The  color  is  too  brown  in  the  plate ;  should  have 
been  white. 

IV.  Agar  stick  culture,  six  days  at  37°.     The  puncture 

does  not  always  turn  out  so  vigorous. 
V.  Agar  stick  culture,  six  days  at  37°.     Surface. 
VI.  Agar  plate,  five  days  at  37°.     Natural  size. 
VII.  Gelatin  plate,  eight  days  at  22°.     In  nature  the 

colonies  are  pure  white.     Natural  size. 
VIII.   Gelatin  plate,  eight  days  at  22°.     x60.    The  larger 
colony  is  superficial,  the  smaller  ones  are  deep. 
IX.  Microscopical  preparation  from  an  agar   culture 
(x800)  two  days  old.     We  do  not  always  find 
tetrads  alone.     There  are  numerous  individual 
cocci. 
X.  Potato  culture,  seven  days  at  37°. 
XI.  Microscopical  appearances.     Tetrads  before,  dur- 
ing, and  after  division  highly  magnified. 


XI. 


Explanation  of  Plate  8. 

Micrococcus  LUTEus.     Cohen  with  Lehm.  andNeum. 
I.   Gelatin  stick  culture,  six  days  Sit  22°. 
II.  Gelatin  plate,  three  days  at  22°.      x  50.     On  right 
side  superficial,  on  left  side  deep-seated  colony. 

III.  Microscopical  preparation  (x  1,000)  from  an  agar 

plate  two  days  old.     The  micrococci  are  often 
aggregated  into  tetrads. 

IV.  Agar    plate    (natural   size),    five    days    at    22°. 

The  colonies  are  sometimes  more  yellow. 
V.  Potato  culture,  six  days  at  22°.     Sometimes,  has  a 
dull  lustre. 

Sarcina  pulmonum.     Virchow,   Hauser. 

VI.   Gelatin   stick  culture,  twenty  days  at  22°.     In 
reality  the  puncture  is  grayer  in  color. 
VII.  Agar  streak,  twenty  days  at  22°. 
VIII.  Gelatin  plate,  twenty  days  at  22°.     On  the  right, 
superficial  colony ;  on  the  left,  deep-seated  one. 
IX.  Potato  culture,  twenty  days  at  22°. 


Tab.     8. 


Tab.     9. 


Explanation  of  Plate  9. 

Sakcina  flava.    De  Bary  with  Lehm.  and  Stubenrath. 

I.  Gelatin  stick  culture,  ten  days  at  22°. 
II.   Agar  streak  culture,  six  days  at  22°. 

III.  Agar  stick  culture,   six  days  at  22°.     Puncture 

canal. 

IV.  Agar  stick  culture,  six  days  at  22°.     Surface. 
V.   Gelatin  plate,  five  days  at  22°.     Natural  size. 

VI.  Gelatin  plate,  five  days  at  22°.     x60.     Superficial 
colony. 
VII.  Agar  plate,  six  days  at  22°.     Natural  size. 
VIII.  Agar  plate,  six  days  at  22°.     x60.     Upper  colony 
superficial,  lower  colony  deep  seated. 
IX.  Potato  culture,  ten  days  at  22°. 
X;  Microscopical  preparation.     Pure  culture  from  an 
agar  plate,     x  1?  000.     Stained  with  f uchsin  and 
decolorized  with  acetic  acid. 
XI.  Microscopical    preparation.     Pure    culture   from 
bouillon.     Unstained.      xljOOO. 
XII.  Sarcina  in  the  shape  of  bales  (regular  combination 

of  individual  packages). 
XIII.   Sarcina  in  heaps  of  packages  (irregular  mass  of 
single  regular  or  irregular  packages). 


xn.  XIII. 


Explanation    of    Plate     10. 

Sarcina  aurantiaca.     riiigge. 

I.  Gelatin  stick  culture,  six  days  at  22°. 
II.  Agar  streak  culture,  five  days  at  22°.     The  color 
is  not  so  red  in  all  cases,  usually  it  is  a  bright 
orange.     Likewise  in  the  agar  stick  and  potato 
cultures. 

III.  Agar   stick  culture,  six  days  at  22°.     Puncture 

canal. 

IV.  Agar  stick  culture,  six  days  at  22°.     Surface. 

V.  Gelatin  plate,  five   days  at  22°.     Natural   size. 
The  gray  rim  around  the  colony  indicates  the 
depression. 
VI.  Gelatin  plate,  five  days  at  22°.      x60.     A  young 
colony.     The  gray  ring  indicates  the  zone  of 
depression. 
VII.  Agar  plate,  five  days  at  22°.     Natural  size. 
VIII.  Agar  plate,  five  days  at  22°.     x  60.     Upper  colony 
superficial,   lower  colonies  deep  seated.     The 
superficial  colonies  are  usually  opaque  toward 
the  middle. 
IX.  Potato  culture  eight  days  old. 
X.  Microscopical  preparation.     Pure  culture  of  agar. 
X  1,000.     Colored  with  fuchsin  and   decolor- 
ized with  acetic  acid. 
XI.  Microscopical    preparation.     Pure   culture    from 
bouillon.      xl?O0O.      Unstained.      Semi-sche- 
matic. 


10 


Tab.  10. 


i. — - 


V 


1 

tf 

Lz 

'  vu- 

IX 


Tab.    11 


Explanation  of  Plate  11. 

Sarcin^   Diverse. 

I.  Sarcina  cervina  Stubenrath.     Agar  streak  culture, 
fifteen  days  at  22'',  isolated  from  gastric  con- 
tents. 
II.   Sarcina   pulmonum   Virchow.     Agar   streak  cul- 
ture, fifteen  days  at  37°. 

III.  Sarcina  erythromyxa  Krai.     Agar  streak  culture, 

thirty  days  at  22°,  isolated  from  beer. 

IV.  Sarcina  lutea  Fliigge.     Agar  streak  culture,  ten 

days  at  22°,  isolated  from  stomach. 
V.   Sarcina  aurantiaca  Fliigge.     Agar  streak  culture, 

ten  days  at  22°,  isolated  from  dough. 
VI.     Sarcina  rosea  Schroeter  and  Zimmermann.  Agar 
streak  culture,  twenty-five  days  at  22°,  isolated 
from  "  weissbeer. " 
VII.   Micrococcus  badius  Lehman  and  Neumann.     Agar 
streak  culture,  fifteen  days  at  22°,  isolated  from 
the  atmosphere. 
VIII.   Sarcina  canescens  Stubenrath.     Agar  streak  cul- 
ture, twenty  days  at  22""^  isolated  from  stomach. 


11 


Explanation  of  Plate  12. 

Bacterium  pneumonia.     Friedlander. 

(Friedlander's  pneumonia  bacillus.) 

I.  Agar  streak  culture,  four  days  at  22°. 
II.  Gelatin  stick  culture,  ten  days  at  22°. 

III.  Agar  stick  culture,  four  days  at  22°.     Puncture 

canal. 

IV.  Agar  stick  culture,  four  days  at  22°.     Surface. 
V.  Gelatin  plate,  three  days  at  22°.     Natural  size. 

VI.  Agar  plate,  two  days  at  22°.      x60.     The  brown, 

whetstone-shaped  colony  is  deep  seated. 
VII.  Gelatin  plate,  three  days  at  22°.      x  50.     Above, 

superficial  colony ;  below,  deep-seated  one. 
VIII.  Agar  plate,  four  days  at  22°.     Natural  size.  The 

delicate  gray  colonies  and  the  smallest  ones  are 

deep  seated.     One  colony  has  been  colored  too 

yellow. 
IX.   Microscopical  preparation.     Pure  culture  ( x  800) 

from  an  agar  plate.     Stained  with  fuchsin. 
X.  Microscopical    preparation.      Smear    preparation 

from  sputum.      X  800.     Fuchsin  stain. 
XI.  Potato  culture,  six  days. 


13 


Tab.   12. 


Tab.  13. 


Explanation  of  Plate  13. 

Bacterium  acidi  lactici.     Fltigge. 

(Lactic-acid  bacillus. ) 

I.   Gelatin  stick  culture,  five  days  at  22°.     In  nature 
the  puncture  canal  is  a  little  whiter. 
II.  Agar  streak  culture,  five  days  at  22°. 

III.  Agar  stick  culture,  three  days  at  22°.     Puncture 

canal. 

IV.  Agar  stick  culture,  three  days  at  22°.     Surface. 
V.  Agar  plate,  three  days  at  22°.     Natural  size. 

VI.  Agar  plate,  three  days  at  22°.      x  50.     Upper  col- 
ony superficial,  lower  ones  deep  seated.      Vide 
PI.  14,  VII. 
VII.   Gelatin  plate,  two  days  at  22°. 
VIII.  Gelatin  plate,  two  days  at  22°.     x50.     Upper  col- 
ony   superficial,    lower    colonies   deep   seated. 
The  superficial  colony  may  vary  extremely  in 
its  growth.      Vide  PL  15,  IV.,  VII. ;    PI.  16, 
IX.,  VIII.;  PI.  17,  I.,  II. 
IX.   Microscopical  preparation.     Pure  culture  from  an 

agar  colony,      x  800. 
X.  Potato  culture,  six  days  at  22°.     The  air  bubbles 
on  the  surface  often  cover  it  completely. 


13 


Explanation  of  Plate  14. 

Bacterium  coli  commune.     Escherich. 

I.   Gelatin  stick  culture,  ten  days  at  22°. 
II.  Gelatin  streak  culture,  four  days  at  22°.     In  na- 
ture, transparent  and  iridescent  like  mother-of- 
pearl.      VideTl.  16,  VI. 

III.  Agar  streak  culture,  four  days  at  22°.      Vide  PI. 

16,  V. 

IV.  Agar  stick  culture,  two  days  at  22°.     Puncture 

canal. 
V.  Agar  stick  culture,  two  days  at  22°.     Surface. 

VI.  Agar  plate,  four  days  at  22°.      x60.     Deep-seated 
colonies.      VideVL  13,  VI. 

VII.  Agar  plate,  four  days  at  22°.  x60.  A  part  of  a 
superficial  colony.  During  growth  occasionally 
exhibits  forms  like  bacillus  acidi  lactici.  Vide 
PI.  13,  VI. ;  PI.  17,  v.,  VI. ;  PI.  18,  IV. ;  PI. 
12,  VIII. 
VIII.  Agar  plate,  three  days  at  22°.     Natural  size. 

IX.  Potato  culture,  five  days  at  22°.     May  also  ap- 
pear of  a  lighter  or  darker  color. 
X.  Bacteria  with  long  flagella  from  bacterium  bras- 
sicse  acidse.       xljOOO.      Stained  according  to 
Loffler's  method. 

XI.  Bacteria  with  flagella,  from  the  bacterium  of  pig- 
eon diphtheria.  xljOOO.  Stained  by  Loffler's 
method. 
XII.  Bacteria  with  one  flagellum,  rarely  with  two  fla- 
gella, from  bacterium  of  the  deer  plague. 
X  1,000.     Stained  by  Loffler's  method. 


Tab.   14. 


viu. 


Tab.  15. 


VTl 


VTII 


Explanation  of  Plate  15. 

Bacterium  coli  commune.     Escherich. 

I.  Gelatin  plate,  eight  days  at  22°.     x60.     Coli  culti- 
vated from  pus.     Deep-seated  colonies.     Ab- 
normal shapes. 
II.   Gelatin  j)late,  four  days  at  22°.     Natural  size. 

III.  Gelatin  plate,  one  day  at  22°.      x  90.     Superficial 

colony.      Vide  PL  13,  VIII. ;  PL  16,  VIII. 

IV.  Gelatin  plate,  four  days  at  22°.     x  60.     Superficial 

colony.      Vide  PL  16,  IX. ;  PL  17,  I.,  II. 
V.  Gelatin  plate,   four  days  at  22°.      x60.     Deep- 
seated  colony. 
VI.  Gelatin  plate,  ten  days  at  22°.      x90.     Superficial 

colony. 
VII.  Gelatin  plate,  ten  days  at  22°.     x90.     Superficial 
colony. 
VIII.  Microscopical  preparation.     Pure  culture  from  an 
agar  plate,      x  500. 
IX.  Bacteria  of  various  kinds  of  coli.     x  1,000.    Great 
differences  in  size. 


•  /•   V 

IX. 


16 


Explanation  of  Plate  16. 

Bacterium  typhi.     Eberth,   Gaffky. 

(Typhoid  bacillus.) 

I.  Agar  stick  culture,  three  days  at  22°.     Puncture 

canal. 
II.  Agar  stick  culture,  three  days  at  22°.     Surface. 

III.  Gelatin  stick  culture,  eight  days  at  22°.     Punc- 

ture canal. 

IV.  Gelatin  stick  culture,  eight  days  at  22°.     Surface. 
y.  Agar  streak  culture,  four  days  at  22°.      Vide  PI. 

14,  III. 
VI.  Gelatin  streak  culture,  three  days  at  22°.      Vide 

PI.  14,  II. 
VII.   Gelatin  plate,  one  and  a  half  days  at  22°.     Deep- 
seated  colony.      Vide  PI.  15,  V. ;  PI.  13,  VIII. 
VIII.  Gelatin  plate,  one  and  a  half  days  at  22'^'.     Super- 
ficial colony.      Vide  PI.  15,  III. ;  PI.  13,  VIII. 
IX.   Gelatin  plate,  four  days  at  22°.     Superficial  col- 
ony.    Vide  PI.  15,  IV.,  VII. 


16 


Tab.   16. 


M 


va 


□  □ 


L\. 


VTU 


Tab.  17. 


Explanation  of  Plate  17. 

Bacterium  typhi.     Eberth,   Gaffky. 

(Typhoid  bacillus.) 

I.  Gelatin  plate,  eight  days  at  22°.      x90.     Superfi- 
cial colony.      Vide  PI.  15,  VII.,  VI. 
II.  Gelatin  plate,   eight  days  at  22°.      xl50.     Su- 
perficial colony. 

III.  Gelatin  plate,  four  days  at  22°.     Natural  size. 

IV.  Agar  plate,  four  days  at  22°.     Natural  size. 

V.  Agar  plate,  four  days  at  22°.      x60.     Deep-seated 

colonies. 
VI.  Agar  plate,  four  days  at  22°.      x60.     Superficial 
colonies. 
VII.  Potato  culture,  five  days  at  22°. 
VIII.  Microscopical    preparation.     Pure    culture    from 
agar  plate,      x  1,000. 
IX.  Microscopical  preparation.     Bacilli  with  flagella. 
Copied  from  Fraenkel  and  Pf eiffer :  "  Atlas  d. 
Bakterienkunde,"  Plate  54,  Fig.  111. 
X.  Microscopical  preparation.     Long  thread,  thickly 
studded  with  flagella.    x  1, 500.    Loffier'  s  stain. 
XI.  Microscopical    preparation    of    bacterium    typhi 
murium    Loftier,   with    flagella    and    capsule. 
X  1,500.     Stained  by  Loffler's  method. 


17 


Explanation  of  Plate  18. 

Bacterium  sEPTic^MiiE  hemorrhagica.     Htippe. 

(Fowl  cholera,  rabbit  septicaemia,  etc.) 

I.  Gelatin  stick  culture,  seven  days  at  22°. 

II.  Agar  streak  culture,  seven  days  at  22°. 

III.  Agar  plate,  five  days  at  22°.     Natural  size. 

IV.  Agar  plate,  five  days  at  22°.      x60.     Superficial 

colony.      Vide  PI.  17,  YI. ;  14,  YII. ;  13,  VI. 
V.  Agar  plate,  five  days  at  22°.     x60.     Deep-seated 
colonies. 
VI.  Gelatin  plate,  five  days  at  22°.     Natural  size. 
VII.  Gelatin  plate,  five  days  at  22°.    x90.  Deep-seated 
colonies. 
VIII.  Gelatin  plate,  five  days  at  22°.     x  90.    Superficial 
colony.      Vide  PL  17,  I.;    PL  16,  IV.,  VIII.; 
PL  15,  IV.,  III.,  VII. ;  PL  13,  VIII. 
IX.  Microscopical  preparation,    x  1,000.    Pure  culture 
from  an  agar  plate. 
X.  Individual   bacteria.     Highly   magnified.     Sche- 
matic. 


18 


Tab.  18. 


Tab.   19. 


Explanation  of  Plate  19. 

Bacterium  mallei.     Loffler. 

(Glanders.) 

I.  Gelatin  stick  culture,  six  days  at  22°. 
II.  Agar  streak  culture,  six  days  at  37°.     The  mid- 
dle white  line  is  not  always  so  pronounced. 

III.  Agar  stick  culture,  three  days  at  37°.     Puncture 

canal. 

IV.  Agar  stick  culture,  three  days  at  37°.     Surface. 
V.   Gelatin  plate,  five  days  at  22°.     Natural  size. 

VI.  Gelatin  plate,  four  days  at  22°.      x60.     Upper 

colony  superficial,  lower  colonies  deep  seated. 
VII.  Agar  plate,  two  days  at  22°.      x60.     Upper  col- 
ony superficial,  lower  colonies  deep  seated. 
VIII.  Microscopical  preparation.    Pure  culture.     X  800. 
Puchsin  stain. 
IX.  Potato  culture,  two  days  at  37°. 
X.  Potato  culture,  twenty  days  at  37°. 
XI.  Individual  bacteria.    Highly  magnified.    In  some 
places  the  staining  fluid  is  absorbed  poorly  or 
not  at  all. 


XI. 


X» 


Explanation  of  Plate  20. 

CoRYNEBACTERiuM    DIPHTHERIA.      (Lo£9.er)    Lehmann 
and  Neumann. 

(Diphtheria  bacillus.) 

I.  Glycerin-agar  stick  culture,  twenty  days  at  22°. 

Puncture  canal. 
II.   Glycerin-agar  streak  culture,  eight  days  at  22°. 
III.  Glycerin-agar  stick  culture,  twenty  days  at  22°. 

Surface. 
lY.  Glycerin-agar  plate,   eight  days   at  22°.      x60. 

Deep  and  superficial  colonies. 
V.  Glycerin-agar  plate,  forty  days  at  22°.     x  60.     On 
the  left  side  deep-seated  colonies ;  on  the  right 
side  deep  and  superficial  colonies. 
VI.   Glycerin-gelatin    plate,    twenty    days     at     22°. 

Natural  size.     Superficial  and  deep  colonies. 
VII.  Glycerin-gelatin  plate,  twenty  days  at  22°.     x60. 
On  the  left  side  deep-seated  colonies;    on  the 
right  side  superficial  ones. 
VIII.  Potato  culture,  fourteen  days  at  22°. 
IX.  Microscopical    preparation.     Pure    culture    from 

bouillon  two  days  old.      x  TOO. 
X.  Microscopical   preparation.      Pure   culture   from 
bouillon.     Involution  forms.     About  x  1,200. 
XI.  Individual   bacteria.     Highly   magnified.     Sche- 
matic. 


20 


Tab.  20. 


ua^ 


vm 


a 


Tab.  21 


YE. 


Vffl. 

LitliJ\nxt.y.  F.  ReichlioUi ,  Miiiichf 


Explanation  of  Plate  21. 

Bacterium  latericium.     Adametz. 

I.  Agar  streak  culture,  seven  days  at  22°. 
II.   Gelatin  stick  culture,  fourteen  days  at  22°. 

III.  Gelatin  plate,  seven  days  at  ^l"" .      x60.     Deep- 

seated  colonies  on  the  right,  superficial  on  the 
left. 

IV.  Potato  culture,  thirty  days  at  22°.     Natural  size. 
V.  Agar  plate,  seven  days  at  22°.     Superficial  colony 

on  the  right,  deep  one  on  the  left. 
VI.  Microscopical    preparation.     Pure    culture    from 
agar  twenty-four  hours.     About  x  800. 

Bacterium    h^morrhagicum.       (Kolb)     Lehm.     and 
Neum.      (Morbus  Werlhofii.) 

VII.  Microscopical    preparation.     Pure    culture   from 
bouillon  three  days  old.     (Copied  from  Kolb :  A. 
G.,  Vol.  VII.,  PI.  II.,  Figs,  1  and  2). 
VIII.  Smear  preparation  from  the  liver  of  a  dog.    (Copied 
from  Kolb:  Lo.,  Vol.  VIL,  PL  in.,  Fig.  8.) 


21 


Explanation  of  Plate  22. 

Bacterium  putidum  (Fliigge)  Lehm.  and  Neum. 

(Bacterium  fluorescens  non-liquef aciens  Autor. ) 

I.  Gelatin  stick  culture,  three  days  at  22°. 
II.  Gelatin  plate,  twenty -four  hours  at  22"".      x90. 
Deep-seated  colony. 

III.  Gelatin  plate,  twenty -four  hours  at  22°.      x90. 

Superficial  colony.      Vide  PI.  13,  VIII. ;  PL  15, 
III. 

IV.  Gelatin  plate,  four  days  at  22°.     Natural   size. 

Appearance  of  colonies  upon  a  dark  background. 
V.  Potato  culture,  four  days  at  22"".     Natural  size. 

Vide  PI.  14,  IX. 
VI.  Microscopical    preparation.     Pure    culture    from 
gelatin  plate,      x  800.     Ordinarily  threads  are 
formed  on  agar. 
VII.  Agar   plate,   eight   days   at   22°.     Natural   size. 
Appearance  of  the  colony  on  a  white   back- 
ground. 
VIII.  Agar  plate,  three  days  at  22°.      x60. 
IX.  Bacteria  with  one  flagellum,  more  rarely  two  fla- 
gella.     X  1,000.     Stained  according  to  Loffler's 
method. 


J^ 


EL 


23 


Tab.  22. 


Lithj\iist,v.  ¥  Reiohhold .  Miinrhwi 


Tab.  23. 


p 

w 

''«s  '^' 

[ 

vn. 

ItthJtaslv.  F  Reichhold .  Miinohen 


Explanation  of  Plate  23. 

Bacterium  syncyaneum.    (Ehrenb.)  Lehm.  and  Neum. 

(Bacillus  cyanogenes  Fliigge ;  blue  milk. ) 

I. -III.   Gelatin  stick  cultures,  six  to  ten  days  at  22°. 
Other  shades  of  color  are  also  observed. 
IV.  Agar  stick  culture,  ten  days  at  37°. 
V.  Bouillon  culture,  four  days  at  37°. 
VI.  Milk  culture,  three  days  at  37°.  upon  non-steril- 
ized milk. 
VII.  Microscopical   preparation.     Pure    culture    from 

agar  plate,     x  800. 
VIII.  Microscopical   preparation.     Pure  culture.     Fla- 
gella  stained  with  Loffler^s  mordant. 
IX.  Bacteria  with  flagella ;  one  or  more  at  a  pole,      x 
1,000.     Stained  by  Loffler's  method. 


H 


IX. 


23 


Explanation  of  Plate  24. 

Bacterium  syncyaneum.    (Ehrenb.)  Lelun.  and  Neum. 

(Bacillus  cyanogenes  Fliigge ;  blue  milk. ) 

I.-III.  Potato  cultures,  three  to  ten  days  at  22°. 

Potatoes  of  different  kinds  inoculated  with  the 

same  culture.     The  differences  in  color  may  be 

still  more  manifold. 

IV.  Agar  plate,  three  days  at  22°.     Natural  size. 

V.  Agar  plate,  three  days  at  22° .    x  60.     On  the  right 

deep-seated,  on  the  left  superficial  colonies. 
VI.  Gelatin  plate,  three  days  at  22°.     Natural  size. 
VII.  Gelatin  plate,  eight  days  at  22°.     Natural  size. 
View  of   the   colonies   against  a  white   back- 
ground. 
VIII.  Gelatin  plate,  three  days  at  22°.      x60.     Above, 
superficial  j  below,  deep-seated  colonies. 


34 


Tab.  24. 


\TiT 

Aiisi  \  y  i;i'irhhol(i.Miinfhi'i 


Tab.  25. 


!;.  ;.l,!'<v|,|  M„...|.cM 


Explanation  of  Plate  25. 

Bacterium  prodigiosum.    (Ehrenb.)  Lehm.  and  Neum. 

I.  Gelatin  stick  culture,  one  day  at  22". 
II.  Agar  streak  culture,  four  days  at  22"^. 

III.  Agar  stick  culture,  four  days  at  22°.     Puncture 

canal. 

IV.  Agar  stick  culture,  four  days  at  22°.     Surface. 
V.  Agar  plate,  two  to  four  days  at  22°.     Natural 

size.     Colonies  with,  and  without  development 
of  coloring  matter. 
VI.  Agar  plate,  eight  days  at  22°.      x  60.     Superficial 
colonies  reddish,  deep  ones  yellowish. 
VII.   Gelatin  plate,  two  days  at  22°.     x60.     Superficial 
colony  just  beginning  to  sink. 
VIII.   Gelatin  plate,  two  days  at  22°.     Natural  size. 
IX.  Potato  culture,  eight  days  at  22°.     Typical,  with 

metallic  reflex  on  the  surface. 
X.  Potato  culture,  eight  days  at  22°.     Atypical,  white 
deposit. 
XI.  Microscopical    preparation.     Pure   culture    from 
agar.      x800.     Fuchsin  stain. 
XII.  Bacteria  with  several  flagella.      x  1?  000.     Stained 
by  Lofiler's  method. 


XII. 


25 


Explanation  of  Plate  26. 

Bacterium  kiliense.     (Breunig   and   Fischer)  Lehin. 
and  Neum. 

(Kiel water  bacillus.) 

I.  Agar  streak  culture,  four  days  at  22'^. 
II.   Gelatin  stick  culture,  four  days  at  22"".     Colony 
without  development  of  coloring  matter. 

III.  Gelatin  plate,    five  days  at  l^"".     Natural  size. 

Colonies  with  and  without  development  of  color- 
ing matter. 

IV.  Gelatin  plate,  five  days  at  22"".     x  60.     Superficial 

colony. 
V.  Gelatin  plate,  five  days  at  22°.    x  60.    Deep-seated 
colony. 

VI.  Agar,  plate,  five  days  at  22°.  Natural  size.  Col- 
ored and  uncolored,  superficial  and  deep-seated 
colonies. 

VII.  Agar  plate,  five  days  at  22°.  x60.  Uncolored 
colonies.  On  the  right  side,  superficial;  on 
the  left,  deep  seated. 
VIII.  Agar  plate,  five  days  at  22"".  x  60.  Colored  colo- 
nies. On  the  right,  superficial;  on  the  left, 
deep  seated. 

IX.  Microscopical  preparation,      x  1,000.     Pure  cul- 
ture from  agar  plate.     Fuchsin  stain. 
X.  Potato  culture,  five  days  at  22°. 

XI.  Bacteria  with  several  flagella.  x  1,000.  Stained 
by  Loffler's  method. 

r 

XI. 

26 


Tab.  26, 


Tab.  27. 


Explanation    of    Plate    27. 

Bacterium  janthinum.     Zopf. 

I.  Gelatin  stick  culture,  ten  days  at  ordinary  tem- 
perature. 
II.   Agar  streak  culture,  six  days  at  ordinary  tempera- 
ture.      The  white   borders  at  the   sides   also 
become  violet  after  prolonged  standing. 

III.  Agar  stick  culture,  seven  days  at  ordinary  tem- 

perature.    Puncture  canal. 

IV.  Agar  stick  culture,  seven  days  at  ordinary  tem- 

perature.    Surface. 
V.  Agar  plate  culture  (x60),  four  days  at  ordinary 
temperature.      Superficial    and    deep    colony. 
Within  the  former  the  original  colony  is  still 
visible. 
VI.  Agar  plate  culture,  eight  days  at  ordinary  tem 
perature.     Natural   size.     The   colonies   often 
take  on  a  dark  violet  color. 
VII.  Gelatin  plate  culture,  six  days  at  ordinary  tem- 
perature.    Natural  size.     The  blue  zones  are 
not  always  so  deeply  colored. 
VIII.   Gelatin  plate  culture,  six  days  at  ordinary  tem- 
perature.     X  60.     The  smaller  colony  is  near 
the  surface,  the  larger  one  upon  the  surface. 
IX.  Microscopical  preparation,  from  a  five  days'  agar 

culture.      X  TOO. 
X.  Potato  culture,  six  days  at  ordinary  temperature. 
XI.  Bacteria  with  flagella.     x  1,000.     Loffler's  stain. 
XII.  Bacteria  with  flagella,  from   a  culture    obtained 
from  Sweden,     xl;000. 


% 


XL  XIL 

27 


Explanation  of  Plate  28. 

Bacterium  fluorescens.     Fltlgge. 

(Bacillus  fluorescens  liquefaciens.     Fltigge.) 

I.  Gelatin  stick  culture,  two  days  at  22°. 

II.  Gelatin  stick  culture,  eight  days  at  22°. 

III.  Agar  streak  culture,  three  days  at  22°. 

IV.  Agar  stick  culture,  four  days  at  22°. 

V.   Gelatin  plate,  two  days  at  22°,    Part  of  a  super- 
ficial colony.     X  90. 
VI.  Agar  plate,  twenty -four  hours  at  22°.      x60.     e, 
superficial;  i,  deep-seated  colony. 
VII.  Gelatin  plate,  three  days  at  22°.     Natural  size. 
VIII.  Microscopical    preparation.     Pure    culture    from 
agar  plate,      x  800. 
IX.  Potato  culture,  four  days  at  22°.      Natural  size. 
Vide  PI.  22,  V. ;  PI.  14,  IX. 
X.  Bacteria  with  flagella,  usually  one,  more  rarely 
two  or  more,     x  1,000.     Loffler's  stain. 


X 


28 


Tab.  28. 


Tab.  29. 


Explanation  of  Plate  29. 

Bacterium  pyocyaneum.     (Fliigge)  Lehm.  and  Neum. 

(Green  pus.) 

I.  Gelatin  stick  culture,  three  days  at  22°. 
II.  Agar  streak  culture,  two  days  at  37°. 

III.  Gelatin   plate,  two  days  at  2'!''.      x60.     Deep- 

seated  colonies  and  some  immediately  beneath 
the  surface,  in  younger  and  older  stages. 

IV.  Gelatin  plate,  five  days  at  22"".      x  60.     Part  of  a 

superficial  colony. 
V.   Gelatin  plate,  two  days  at  22°.     Natural  size. 
VI.  Agar  plate,  two  days  at  37°.     Natural  size. 
VII.  Agar  plate,  two  days  at  37°.     x60.     Below,  deep- 
seated  ;  above,  superficial  colonies. 
VIII.  Potato  culture,  three  days  at  37°.     Natural  size. 
IX.  Microscopical    preparation.     Pure    culture   from 
agar  plate,      x  800. 
X.  Bacteria  with  one,  more  rarely  two  polar  flagella. 
Xl,000.     Loffler's  stain. 


29 


Explanation  of  Plate  30. 

Bacterium  zopfii.     Kurth. 

I.  Gelatin  stick  culture,  six  days  at  22°. 
II.   Gelatin  streak  culture,  thirty-six  hours  at  37°. 
In  reality  of  a  gray  transparent  color. 
III.  Agar  stick  culture,  six  days  at  22°.     Puncture. 
IV.  Agar  stick  culture,  six  days  at  22°.     Surface. 

V.  Gelatin  plate,  seven  days  at  22°.     Natural  size. 
VI.  Gelatin  plate,  thirty-six  hours  at  22°.     Natural 

size. 
VII.  Gelatin  plate,  twenty-four  hours  at  22°:  x  90. 
Thread-like  part  of  the  colony.  Deeply  situ- 
ated. 
VIII.  Gelatin  plate,  twenty-four  hours  at  22°.  x60. 
Superficial  colony.  Vide  PI.  32,  VIII.  5  PL 
33,  VII. 


30 


Tab.  30. 


vni 


VTl. 


Tab    31 


Explanation  of  Plate  31. 

Bacterium  zopfii.    Kurth. 

I.  Gelatin  plate,  eight  days  at  22°.      x90.     Border 

of  a  colony. 
II.  Microscopical  preparation.      xl?000.      Pure  cul- 
ture from  agar  plate.     Stained  with  fuchsin. 

III.  Agar   plate,  twenty -four   hours   at  37".       x60. 

Superficial  colony  surrounded  by  innumerable 
bacteria  which  have  wandered  away. 

IV.  Agar  plate,  twenty -four  hours  at  37°.     Natural 

size. 
V.  Agar  plate,  twelve  hours  at  37°.      Deep-seated 

and  superficial  colony. 
VI.  Agar  plate,  four  days  at  22°.     Deep-seated  colony. 
VII.  Gelatin  plate,  eight  days  at  22°.     Sausage -shaped 

forms  of  a  deep-seated  colony. 
VII.  Bacteria  with  numerous  flagella.     xljOOO.    Loff- 
ler's  stain. 


81 


Explanation  of  Plate  32. 

Bacterium   vulgare   /5   mirabilis.      (Hauser)   Lehm. 
and  Neum. 

(Proteus  mirabilis  Hauser.) 

I.  Agar  stick  culture,  two  days  at  22°.     Puncture 

canal. 
II.  Agar  stick  culture,  two  days  at  22"".     Surface. 

III.  Gelatin  stick  culture,  six  days  at  22°. 

IV.  Agar  streak  culture,  two  days  at  22''. 

V.  Agar  plate,  seven  days  at  22°.     Natural  size. 
VI.  Agar  plate,  seven  days  at  22^^.      x60.     Above, 
superficial;  below,  deep-seated  colony. 
VII.  Gelatin   plate,  two  days  at    Y,22' 
seated  colonies. 
VIII.  Gelatin  plate,  two  days  at  22°. 
ficial  colony. 
IX.  Potato  culture,  eight  days  Sit22°. 
X.  Microscopical   preparation.     Pure 
two  days  old.      x800. 


.     60. 

Deep- 

X60. 

Super- 

Natural  size. 

agar 

culture, 

32 


Tab.  32. 


lu 


vni. 


LuluAnst  y.  Y  Rcirhhold .  Miinrheii 


Tab.  33. 


Explanation  of  Plate  33. 

Bacterium  vulgare  Lehm.  and  Neum, 

(Proteus  vulgaris  Hauser.) 

I.  Gelatin  stick  culture,  twenty-four  hours  at  22°. 
II.  Agar  streak  culture,  thirty -six  hours  at  22°. 

III.  Agar  plate,  thirty-six  hours  at  22°.     Natural  size. 

IV.  Agar   plate,  four   days   at  22°.     x60.     Above, 

superficial ;  below,  deep-seated  culture. 
V.  Gelatin  plate,  thirty -six  hours  at  22°.     Natural 
size. 
VI.   Gelatin    plate,  thirty-six   hours    at   22°.      x60. 
Eight  side,   superficial  j    left  side,  deep-seated 
colonies.     The  lower  one,  emerging  on  the  sur- 
face, is  beginning  to  liquefy. 
VII.  Gelatin  plate,  three  days  at  22°.     x60.     Deep- 
seated  colony.     Zoogloea  form,  like  bacterium 
Zopfii. 
VIII.  Microscopical  preparation,    x  800.    Pure  agar  cul- 
ture.    Fuchsin  stain. 
IX,  Bacteria  with  numerous  flagella.      x  1^000. 


33 


Explanation  of  Plate  34. 

Bacterium  erysipelatos  suum.     Migula. 
(Hog  erysipelas.) 
I.  Gelatin  stick  culture,  five  days  at  22°. 

Bacterium  murisepticum.     Migula. 
(Mouse  septicaemia.) 

II.  Agar  streak  culture,  four  days  at  22°. 

III.  Gelatin  stick  culture,  four  days  at  22°. 

IV.  Agar  stick  culture,  four  days  at  22°.     Surface. 
V.  Gelatin  plate,  four  days  at  22°.     Natural  size. 

VI.  Gelatin  plate,  four  days  at  22°.     x60.     Super- 
ficial colony. 
VII.  Agar  plate,  four  days  at  22°.    x60.     Eight  side, 
superficial;  left  side,  deep-seated  colony. 
VIII.  Microscopical    preparation.     Pure    agar    culture, 
two  days,      x  800. 
IX.  Microscopical    preparation.     Smear    preparation 
from  blood  of  a  mouse's  spleen.      x800. 


34 


Tab.  34 


Tab.  35 


VIII. 


Explanation  of  Plate  35. 

Bacillus  megatherium.     De  Bary. 

I.  Gelatin  stick  culture,  twenty -four  hours  at  22°, 
II.  Agar  streak  culture,  three  days  at  22°. 

III.  Gelatin  plate,  thirty-six  hours  at  22°.     Natural 

size. 

IV.  Gelatin  plate,  thirty-six  hours  at  22"".   x  60.    Deep- 

seated  colony. 
V.   Gelatin  plate,  thirty-six  hours  at  22°.    x60.    Su- 
perficial colony. 
VI.  Agar  plate,  four  days  at  22°.     Natural  size. 
VII.  Agar  plate,  one  day  at  22°.     x60.     Eight  side, 
superficial ;  left  side,  deep-seated  colony. 
VIII.  Agar  plate,  four  days  at  22°.     x60.     Eight  side, 
deep-seated*  left  side,  superficial  colony. 
IX.  Potato  culture,  five  days  at  22°.     Natural  size. 
X.  Microscopical    preparation.     Pure   agar   culture. 
X800. 
XI.  Bacilli  with  numerous  flagella.      x  1^000.     Loff- 
ler^s  stain. 


35 


Explanation  of  Plate  36. 

Bacillus  subtilis.     F.  Cohn. 

(Hay  bacillus.) 

I.  Gelatin  stick  culture,  thirty-six  hours  at  22**. 

II.  Gelatin  stick  culture,  eight  days  at  22°. 

III.  Agar  streak  culture,  two  days  at  37°. 

IV.  Agar  stick  culture,  two  days  at  37°.     Puncture 

canal. 
V.  Agar  stick  culture,  two  days  at  37°.     Surface. 
VI.  Agar  plate,  twelve  hours  at  37°.      x60.     Super- 
ficial colony. 
VII.  Agar  plate,  twelve  hours  at  37°,    x60.     Deep- 
seated  colony. 
VIII.  Agar  plate,  twelve  hours  at  37°.     Natural  size. 


36 


Tab.  36. 


Tab.  37 


Explanation  of  Plate  37. 

Bacillus  subtilis.     F.  Cohn. 

(Hay  bacillus.) 

I.  Potato  culture,  seven  days  at  22°. 
II.  Gelatin  plate,  two  days  at  22".    x  60.   Above,  on 
the  right  side,  a  deep-seated  colony.     Below 
it,   a  colony  lies  directly  at  the  surface.     On 
the  left  a  superficial  colony. 
III.  Gelatin  plate,  two  days  at  22°.     Natural  size. 
IV.  Gelatin  plate,  two  days  at  22°.      xlO. 
V.  Microscopical  preparation  ( x  1?  000)  from  an  agar 
colony  three  hours  old  at  37°.     Stained  with 
fuchsin. 
VI.  Microscopical  preparation.     Bacilli  with  flagella. 
(After  Fischer.)     Very  highly  magnified. 
VII.  Microscopical  preparation  (x  1,000)  from  an  agar 
colony  ten  days  old  at  22°.     Contains  spores. 
Unstained. 
VIII.  Microscopical  preparation  (x700)  from   an  agar 
colony  ten  days  old  at  22°.     Double  stain  with 
carbolized  fuchsin  and  methyl  blue. 
IX.  Bacilli  with  numerous  flagella.      x  1?  000.     Loff- 
ler's  stain. 


37 


Explanation  of  Plate  38. 

Bacillus  anthracis.     F.  Cohn  and  E.  Koch. 

(Anthrax. ) 

I.-V.   Gelatin   stick   cultures,    three   days    at   ^^1°, 
Figs.  I.  and  II.  typical,  the  others  atypical. 
VI.  Agar  streak  culture,  two  days  at  22*^. 
VII.  Agar  stick  culture,  five  days  at  22°.     Puncture 

canal. 
VIII.  Agar  stick  culture,   five  days  at  22°.     Surface. 
Atypical. 
IX.  Agar  stick  culture,  five  days  at  22°.     Surface. 
Typical  \  often  has  a  homogeneous  whitish-gray 
color. 


Tab.  38. 


Tab.  39. 


Explanation  of  Plate  39. 
Bacillus  anthracis.     F.  Colin  and  E.  Koch. 


I. 


(Anthrax.) 


Agar  plate,  four  days  at  22°.    x  60.     On  left  side, 
superficial   colony;    on   right   side,    one   lying 
directly   below   the   surface.     Below,  a  deep- 
seated  colony. 
II.  Agar  plate,  four  days  at  22°.    Natural  size. 
III.  Agar  plate,  thirty-six  hours  at  37°.     xl50.    Bor- 
der of  a  streak  culture.     Superficial  colony. 
IV.  Agar  plate,  thirty-six  hours  at  37°.    xl50.    Deep- 
seated  colony. 
V.  Gelatin  plate,  three  days  at  22°.     Natural  size. 
VI.  Gelatin  plate,  three  days  at  22°.     x60.     Super- 
ficial colony,  about  to  sink. 
VII.  Potato  culture,  six  days  at  22°.     Natural  size. 


39 


Explanation  of  Plate  40. 

Bacillus  anthbacis.     F.   Cohn  and  E.  Koch. 

(Anthrax.) 

I.   Smear  preparation  from  the  blood  of  a  mouse's 

spleen,     x  1,000. 
II.  Impression  preparation  from  agar  plate  culture, 
one  day  at  22°.      x  1,000. 

III.  Unstained  preparation  in  hanging  drop  from  bouil- 

lon  culture,  thirty-six   hours  at  37°.     Spores 
beginning  to  drop  out.     x  1,000. 

IV.  Anthrax  threads  from  agar,   thirty-six  hours  at 

37°.     Stained   with  Ziehl's   solution.     Spores 
red,  bacilli  blue,      x  1,000. 
V.  Involution   forms,    five   weeks   old,    from    agar. 

Stick  culture  stained  with  fuchsiuc      x  1,000. 
VI.  Unstained    preparation   in    hanging    drop   from 
bouillon  culture,  eight  hours  at  37°.     Begin- 
ning of  sporulation.      x  1,000. 


40 


Tab.  40. 


1. 


Tab.  41, 


FReJchhoW.Muncberi 


Explanation  of  Plate  41. 

Bacillus  mycoides.     Fltlgge. 

(Eoot  bacillus.) 

I.  Gelatin  stick  culture,  four  days  at  22". 
II.   Gelatin  stick  culture,  fourteen  days  at  22°. 
III.  Agar  streak  culture,  two  days  at  22°. 
IV.  Agar  stick  culture,  eight  days  at  22°.     Puncture 

canal. 
V.  Agar  stick  culture,  eight  days  at  22°.     Surface. 
VI.  Gelatin  plate,  one  day  at  22°.     Natural  size. 
VII.  Agar  plate,  one  day  at  22°.     Natural  size. 
VIII.  Agar  plate,  four  days  at  22°.     Natural  size. 
IX.   Gelatin  plate,  four  days  at  22°.     Natural  size. 
The  colony  is  about  to  sink. 


41 


Explanation  of  Plate  42. 

Bacillus  mycoides.     Flligge. 

(Boot  bacillus.) 

I.  Agar  plate,  one  day  at  22"^.      x20.     Superficial 
and  deep  colony. 
II.  Potato  culture,  seven  days  at  22°.     Natural  size. 
III.  Microscopical    preparation.     Pure   agar    culture, 
twenty -four  hours.    Puchsin  stain,    x  1,000.    A 
few  bacilli  show  spores. 
ly.  Agar  plate,  one  day  at  22°.      x  100.     Superficial 
and  deep  colony. 

Bacillus  butykicus.     Htippe. 

(fiutyric-acid  bacillus.) 

V.  Potato  culture,  three  days  at  22°. 
VI.   Gelatin  plate,  one  day  at  22°.     x60.     Above,  su- 
perficial ;  below,  deep  colony. 
VII.  Gelatin  plate,  one  and  a  half  days  at 22°.      x60. 

Part  of  a  superficial  colony. 
VII.  a.  Flagella  preparation.     xl?000.   Loftier' s  stain. 

Bacillus  vulgatus.     Migula. 

(Bacillus  mesentericus  vulgatus  Pltigge.     Potato 
bacillus.) 

VIII.  Potato  culture,  five  days  at  22°. 
IX.  Potato  culture,  five  days  at  22'^.     Natural  size. 
Both  forms  of  growth  occur. 


VII.  a. 


42 


Tab.  42. 


Tab.  43. 


Explanation  of  Plate  43. 

Bacillus  vulgatus.     Migula. 

(Bacillus  mesentericus  vulgatus  Mtigge.     Potato 
bacillus.) 

I.  Gelatin  stick  culture,  ten  days  at  22°. 

II.  Agar  streak  culture,  ten  days  at  22°. 

III.  Agar  stick  culture,  six  days  at  22°.     Surface. 

IV.  Agar  plate,  six  days  at  22°.     Natural  size. 

V.  Agar  plate,  six  days  at  22°.     x60.     Deep  colony. 
VI.  Agar  plate,   six  days  at  22°.     x  60.     Superficial 
colonies. 
VII.   Gelatin  plate,  eight  days  at  22°.     Natural  size. 
VIII.  Gelatin  plate  eight  days  at  22°.     x60.    Part  of  a 
superficial  colony. 
IX.  Gelatin  plate,  eight  days  at  22°.     xl50.     Part  of 

a  superficial  colony. 
X.  Potato  culture,  five  days  at  22°.     Natural  size. 
XI.  Microscopical    preparation.     Pure    culture    from 
agar,  one  day.      x800.     Fuchsin  stain. 
XII.  Bacilli  with  numerous  flagella.     x  1,000.    Loffler's 
stain. 


XIL 


43 


Explanation  of  Plate  44. 

Bacillus  mesentericus.     Lehm.  and  Neum. 

(Bacillus  mesentericus  fuscus  Fltigge.) 

I.  Gelatin  stick  culture,  two  days  at  22°. 
II.  Agar  streak  culture,  three  days  at  22°. 
III.  Potato  culture,  one  day  at  22°.     Natural  size. 
IV.  Potato  culture,  five  days  at  22°.     Natural  size. 
V.  Agar  plate,  two  days  at  22°.     Natural  size. 
VI.  Agar  stick  culture,  four  days  at  22°.     Surface. 
VII.  Agar  plate,  two  days  at  22°.     x60.    Above,  super- 
ficial colony ;  below,  deep  colony. 
VIII.  Gelatin   plate,    thirty-six    hours    at  22°.      x60. 
Deep  colony. 
IX.  Gelatin   plate,    thirty-six   hours   at  22°.      x60. 
Superficial  colony. 
X.  Gelatin  plate,  two  days  at  22°.     Natural  size. 
XI.  Gelatin  plate,  one  day  at  22°.    x60.    Eight  side, 
deep  colony ;  left  side,  superficial. 
XII.  Microscopical    preparation.     Pure    culture    from 
agar,  two  days.    x800.    Fuchsin  stain.    A  few 
bacilli  with  spores. 
XIII.  Bacilli  with  numerous  flagella.      x  1,000.     Loif- 
ler's  stain. 


f 

XIIL 

44 


Tab.  44. 


LithJnst.v.  F.  Reichhold,  Miiro  ln-i 


Tab.  45. 


Explanation  of  Plate  45. 

Bacillus  tetani.     Nicolaier. 

(Tetanus  bacillus.) 

I.  Sugar-agar  stick  culture,  three  days  at  37°. 
II.  Sugar-gelatin  stick  culture,  six  days  at  22°. 

III.  Sugar-gelatin  plate,   four  days  at  22°.     Natural 

size.     Cultivated  anaerobic. 

IV.  Sugar-gelatin  plate,  four  days  at  22°.     x60.     Su- 

perficial  and    deep    colony.     Cultivated    ana- 
erobic. 
V.  Sugar-agar  plate,  four  days  at  37".     Natural  size. 
Cultivated  anaerobic. 
VI.  Sugar-agar  plate,  four  days  at  37°.     x60.     Super- 
ficial and  deep  colony.     Cultivated  anaerobic. 
VII.  Microscopic     preparation.     Pure     culture     from 
sugar-agar,    three     days     at    37°.       x  1,000. 
Bacilli  with  spores.     ZiehPs  double  stain. 
VIII.   Microscopic     preparation.     Pure     culture     from 
sugar-agar,  two  days  at  37°.     x  1,000.     A  few 
bacilli  with  spores.     Euchsin  stain. 
IX.  Microscopical    preparation.     Pure    culture    from 
sugar-agar,  twenty -four  hours  at  37°.     x  1,000. 
Extremely  long  filaments  with  faintly  colored 
interspaces. 
X.  Microscopical    preparation.     Pure   culture   from 
sugar-agar,  six  days  at  37°.    x  1,000.    Euchsin 
stain.     Long  filaments  and  spore  chains  with 
faintly  colored  interspaces. 


45 


Explanation  of  Plate  46. 

Bacillus  Chauvcei  of  French  Writers. 
(Symptomatic  Anthrax.) 

I.  Sugar-gelatin  stick  culture,  six  days  at  22°. 

II.  Sugar-agar  stick  culture,  three  days  at  37°. 

III.  Sugar-agar  stick  culture,  three  weeks  at  37°. 

IV.  Sugar-agar  plate,  four  days  at  37°.     Natural  size. 

Cultivated  anaerobic. 
V.  Sugar-agar  plate,  four  days  at  37°.    x  60.     Super- 
ficial and  deep  colony.     Cultivated  anaerobic. 
VI.   Sugar-gelatin   plate,  four  days  at  22°.     Natural 

size.     Cultivated  anaerobic. 
VII.   Sugar-gelatin   plate,    four    days   at  22°.      x60. 
Deep  colony.     Cultivated  anaerobic. 
VIII.   Sugar-gelatin  plate,  two  days  at  22°.     xl50.    Part 
of  a  superficial  colony.     Cultivated  anaerobic. 
IX.  Microscopical    preparation.     Pure    culture    from 
sugar-agar,  three   days   at  37°.     Bacilli  with 
spores  and  spores  that  have  fallen  out.    Fuchsin 
stain.     X  1,000. 


46 


Tab.  46. 


Tab.  47. 


VTI 


Explanation  of  Plate  47. 

Bacillus  cedematis  maligni.     Koch. 

(Malignant  oedema.) 

I.  Sugar-agar  stick  culture,  eight  days  at  37°. 
II.  Microscopical  preparation.  Elagella  plait,  x  1,500. 
(Copied  from  G.  Novy :  Zadi.  f.  Hygiene,  Vol. 
XVII.,  PI.  L,  2.) 

III.  Microscopical  preparation.     Bacilli  with  flagella. 

Pure   culture   from   agar,    twenty-four   hours. 
Loffler's  stain,      x  1,000. 

IV.  Sugar-agar  plate,  four  days  at  22*^.      x60.     Part 

of  a  superficial  colony. 
V.  Sugar-agar  plate,  six  days  at  22°.     Natural  size. 
VI.  Microscopical    preparation.     Pure    culture   from 
agar,  two  days  at  37°.     Eods  with  spores.      X 
1,000.     Puchsin  stain. 
VII.  Microscopical    preparation.     Tissue    juice    from 
guinea-pig.     Smear  preparation.     (Copied  from 
Praenkel  and  Pf eiffer :  "  Mikrophotog.  Atlas, " 
Pi.  XXIII.,  46.) 


47 


Explanation  of  Plate  48. 

Mycobacterium    Tuberculosis    (Koch).    Lehm.    and 

Neum. 

(Tubercle  bacillus.) 

I.  Glycerin-agar  streak  culture,  fourteen  days  at  37". 
II.   Glycerin-agar  streak  culture,  forty  days  at  37°. 
III.  Potato  culture,  forty  days  at  37°. 
IV.  Colonies  of  tubercle  bacilli  in  a  blood-serum  cul-- 
ture.     x700.     (Copied  from  Koch:  ''Aetiol.  d. 
Tubercul.      Mittheil.  d.  kais.  Gesundheitsamt," 
Vol.  II.,  PI.  IX.,  44.) 
V.  Culture  on  blood  serum  from  a  piece  of  freshly 
extirpated  scrofulous  gland.    (Copied  as  above. ) 
VI.  Giant   cell   with   radiating   arrangement   of   the 
bacilli.     From  the  cheesy  bronchial  gland  of  a 
case  of  miliary  tuberculosis.     (Copied  as  above, 
PI.  11. ,  9.) 
VII.  Microscopical  preparation.    Pure  culture.    Stained 
by  ZiehPs  method,      x  1, 000. 
VIII.   Branching  of  tubercle  bacilli.     (Copied  from  Hayo 
Bruns:  C.  B.,  XVII.,  No.  23.) 
IX.  Microscopical    preparation.       Sputum.       ZiehPs 
stain.      X  1,000. 
X.  Individual  bacteria.     Highly  magnified. 


i''^ 


^o 


48 


Tab.  48. 


•^.^ 

iS^^ 

» 

i*^  s 

j^^V^?^ 

r-     ^ 

**-  ' 

,e' 

ll.  J 

^^ 

* 

iiJ 

)^., 

,.*-    % 

mi^^ 

& 

^^ 

^^^^ 

Tab    49. 


VI 


Explanation  of  Plate  49. 

ViBKio  CHOLERA.     (Koch)  Buchner. 

(Comma  bacillus.) 

I.  Gelatin  stick  culture,  two  days  at  22°. 
IT.   Gelatin  stick  culture,  seven  days  at  22°. 

III.  Gelatin  stick  culture,  eight  days  at  22°.     Culture 

from  a  case  of  Asiatic  cholera  in  Hanover. 

IV.  Gelatin  stick  culture,  eight  days  at  22°. 
V.  Agar  streak  culture,  eleven  days  at  22°. 

VI.  Agar  stick  culture,  eight  days  at  22°.     Puncture 
canal. 
VII.  Agar  stick  culture,  eight  days  at  ^'T.     Surface. 
VIII.  Agar  plate,  six  days  at  22°.     Natural  size. 
IX.  Agar  plate,  six  days  at  22°.     Culture  from  a  case 
of  Asiatic  cholera  in  Hanover. 


49 


Explanation  of  Plate  50. 

Vibrio  cholera.     (Koch)  Bucliner. 

(Comma  bacillus.) 

T.  Agar  plate,  thirty-six  hours  at  22°.     x60.     Left 

side,  superficial  5  right  side,  deep  colony. 
II.  Agar  plate,  two  days  at  22°.      x60.     Left  side, 
superficial ;  right  side,  deep  colony. 

III.  Agar  plate,  three  days  at  22°.     x60.     Left  side, 

superficial;  right  side,  deep  colony. 

IV.  Agar  plate,  three  weeks  at  22°.     x60.     Leftside, 

superficial ;  right  side,  deep  colony. 
V.  Agar  plate,  five  days  at  22°  ( x  60) ,  from  a  case 
of  Asiatic  cholera  in  Hanover.     Superficial  and 
deep  colony. 
VI.  Gelatin  plate,  four  days  at  22°.     Natural  size. 

Much  depressed  liquefaction  funnel. 
VII.   Gelatin   plate,    fourteen   days   at   22''.     Natural 
size.     Colony  with  pronounced  zonal  develop- 
ment. 
VIII.   Gelatin  plate,  four  days  at  22°.     Shallow  zones 
of  liquefaction. 
IX.   Gelatin  plate,  six  days  at  22°.     Shallow  sunken 
colonies  with  concentric  zones  of  liquefaction. 


50 


Tab.  50. 


nmi 


m. 


iv: 


@     © 
0 


vn 


\i, 


\; 


VTU 


DC. 


Tab.  51. 


Explanation  of  Plate  51. 

ViBBio  CHOLERA.     (Koch)  Buchnei. 

(Comma  bacillus.) 

I.   Gelatin  plate,  thirty-six  hours  at  22°.     x  60.     Su- 
perficial and  deep  colonies. 
II.  Gelatin   plate,   forty-eight  hours  at  22°.      x60. 
Left  side,  superficial ;  right  side,  deep  colonies. 

III.  Gelatin  plate,  three  days  at  22°.      x60.     Super- 

ficial colonies  with  zones  of  liquefaction. 

IV.  Gelatin  plate,  three  days  at  22°.      x60.     Deep 

colony. 
V.   Gelatin  plate,  four  days  at  22°.      x60.     Super- 
ficial colony  with  zone  of  liquefaction. 
VI.  Gelatin   plate,   four  days  at  22°.      x60.     Deep 

colony. 
VII.   Gelatin  plate,  five  days  at  22°.     x60.     Deep  col- 
ony from  a  culture  from  a  case  in  Hanover. 
VIII.  Gelatin  plate,  five  days  at  22°.     x  60.     Superficial 
colony.     Has  undergone  complete  liquefaction. 
IX.   Gelatin  plate,  eight  days  at  22°.      x60.     Super- 
ficial colony  with  zone  of  liquefaction. 


51 


Explanation  of  Plate  52. 

ViBRia  CHOLERA.     (Koch)  Buchner. 

(Comma  bacillus.) 

I.  Gelatin  plate,  five  days  at  22°.  x60.  Abnormal 
shape  of  a  superficial  colony. 

II.  Gelatin  plate,  five  days  at  22".     x90.     Abnormal 

shape  of  a  superficial  colony. 
III.  Gelatin  plate,  five  days  at  22°.      x60.     Deeply 
sunken,  superficial  colony  with  strongly  reflect- 
ing zone  of  liquefaction. 

ly.  Gelatin  plate,  six  days  at  22°.  x60.  Superficial, 
abnormal  colony  with  compact  nucleus.  Shallow 
sinking  in,  with  zone  of  liquefaction. 

V.  Gelatin  plate,  six  days  at  22°.  x60.  Deep,  ab- 
normal colony,  dark,  with  radiating  stripes, 
from  the  same  plate  as  IV. 

VI.   Potato  culture,  two  days  at  22°.     Natural  size. 
Soaked  in  a  solution  of  soda  before  inoculation. 
VII.  Potato  culture,  five  days  at  22°.     Upon  ordinary 
potato. 


52 


Tab.  52. 


Tab.  53. 


m 


Explanation  of  Plate  53. 

Vibrio  cholera  (Koch)  Buchner. 

(Comma  bacillus.) 

I.  Pure  culture  from  bouillon,  twenty-four  hours  at 
37°.     Fuchsin  stain,      x  1, 000. 
II.  Pure  culture  from  agar,  twenty-four  hours,      x 
1,000.     Loffler's  stain  of  flagella. 

III.  Pure  culture  on  gelatin,  forty-eight  hours.     Per- 

fectly fresh  preparation  from  water.     (Copied 
from  Fraenkel  and  Pfeiffer,  Fig.  94.) 

IV.  Pure  agar  culture,  four  weeks  old.     Involution 

forms  stained  with  fuchsin. 
V.  Vibrio  Metschnikovii  Gamaleia.     Smear  prepara- 
tion from  pigeon' s  blood.     (Copied  from  Fraen- 
kel and  Pfeiffer,  Fig.  102.) 
VI.   Vibrio  proteus  Buchner.     Pure  culture  in  bouil- 
lon, twenty- four  hours.     Stained  with  fuchsin. 


53 


Explanation  of  Plate  54. 

Vibrio  albensis.     Lehm.  and  Neum. 

(Fluorescent  Elbe  vibrio.) 

I.   Gelatin  stick  culture,  twenty-four  hours  at  22°. 
II.   Gelatin  stick  culture,  four  days  at  22*^. 

III.  Gelatin  stick  culture,  ten  days  at  22°. 

IV.  Indol  reaction  at  end  of  ten  days.     Bouillon  cul- 

ture treated  with  dilute  sulphuric  acid. 
V.   Gelatin  plate,  three  days  at  22°.      x  60.     Super- 
ficial colony. 
VI.  Gelatin  plate,  three  days  at  22°.      x  60.     Deep 
colony. 
VII.   Gelatin  plate,  thirty-six  hours  at  22°.     Natural 
size. 
VIII.  Microscopical  preparation.     Pure  culture  on  agar, 
forty-eight  hours.     Stained  with  fuchsin. 


54 


Tab.  54. 


Tab.  55. 


Vlll. 


IX, 


Explanation  of  Plate  55. 

Vibrio  danubicus  Heider;  Vibrio  berolinensis 
Rubner;  Vibrioa  quatilis  Gtinther. 

I.  Vibrio   danubicus.     Gelatin   stick   culture,  three 
days  at  22°. 

III.  Vibrio  danubicus.     Gelatin  plate,  three  days  at 

22°.     Right  side,  superficial;    left  side,  deep 
colony. 

IV.  Vibrio    danubicus.      Microscopical    preparation. 

Pure  agar  culture,  twenty-four  hours.     Stained 
with  fuchsin.      x  800. 
V.  Vibrio  berolinensis.     Gelatin  plate,  three  days  at 
22"^.      x60.     Right  side,  superficial ;  left  side, 
deep  colony. 
VI.  Vibrio   berolinensis.     Microscopical   preparation. 
Pure  agar  culture,  twenty -four  hours.     Fuch- 
sin stain.      X  800. 
II.  Vibrio    aquatilis.     Gelatin    stick   culture,    three 
days  at  22°. 
VII.  Vibrio   aquatilis.     Gelatin   plate,  three   days   at 
22°.      x60.     Deep-seated  secondary  colonies, 
sta'rting  from  one  point. 
VIII.  Vibrio     aquatilis.       Microscopical     preparation. 
Pure  agar  culture,  twenty-four  hours.     Fuch- 
sin stain.      X  800. 
IX.  Vibrio  aquatilis.     Gelatin  plate,  three  days  at 
22°.     x60.     Right  side,  superficial;  leftside, 
deep  colonies, 


56 


Explanation  of  Plate  56. 

Vibrio  proteus.     Buchner. 

(Vibrio  Tinkler.) 

I.  Gelatin  stick  culture,  one  day  at  22°. 
II.  Gelatin  stick  culture,  four  days  at  22°. 

III.  Gelatin  plate,  one  day  at  22°.     Natural  size. 

IV.  Gelatin  plate,  four  days  at  22°.      x  60.     Super- 

ficial colony. 
V.  Gelatin   plate,  four   days   at  22^^.     x60.     Deep 
colony. 
VI.  Agar  streak  culture,  six  days  at  22°. 
VII.  Agar  plate,  four  days  at  22°.      x  60.     Superficial 
colony. 
VIII.  Agar  plate,  four  days  at  22"^.    x  60.    Deep  colony. 
IX.  Agar  plate,  four  days  at  22°.     Natural  size. 


56 


Tab    56. 


Tab.  49. 


Explanation  of  Plate  57. 

Spirillum  rubrum.     v.  Esmarch. 

I.  Agar  stick  culture,  ten  days  at  22°, 
II.  Agar  streak  culture,  twenty  days  at  22°. 

III.  Aguv  plate,  five  days  at  22°.      x  60.     e,   Super 

ficial;  if  deep  colony. 

IV.  Gelatin  plate,  seven  days  at  22°.      x  60.     e,  Su- 

perficial ;  i,  deep  colony. 

V.  Microscopical  preparation.  Pure  culture  in  ten- 
fold diluted  bouillon,  two  days  at  37°.  x 
1,000.     Stained  with  fuciisin. 

V.  a,  Flagella  preparation  of  spirillum  rubrum. 
X  1,000,     Loffler's  stain. 


^/i 


V.  a 


Spirillum  ooncentricum.     Kitasato. 

VT.  Agar  plate,  seven  days  at  22°.      x  60.     e.  Super- 
ficial; if  deep  colony. 
VII.   Gelatin  plate,  three  days  at  22°.      x60.     6,  Su- 
perficial; i,  deep  colony. 
VIII.  Agar  plate,  seven  days  at  22°.     Natural  size. 
IX.  Microscopical  preparation.     Pure  culture  in  bouil- 
lon, two  days  at  37°.     x  1,000.    Fuchsin  stain. 


57 


Explanation  of  Plate  68. 
Spirilla. 

I.  Spirillum  serpens  with  plasma  border  which  is 
stained  with  difficulty.  x  1,000.  Fuchsin 
stain.  (Copied  from  Zettnow:  C.  B.,  X.,  PI.  5.) 
II.  Spirilla  from  nasal  mucus.  Smear  preparation. 
X  1,000.  (Copied  from  Weibel:  C.  B.,  TI.,  p. 
468,  Fig.  1.) 

III.  Spirilla  from  nasal  mucus.     Agar  plate,  pure  cul- 

ture.     X  1,000.      (Copied,  as  above,    p.    468, 
Fig.  2.) 

IV.  Spirilla  from  nasal  mucus.     Gelatin  plate,  pure 

culture.      X  1,000.     (Copied,  as  above,  p.  468, 
Fig.  3.) 
V.   Spirillum  uniula  with  flagella.      x  800.     (Copied 
from  Loffler:  C.  B.,  VI.,  PI.  I.,  Fig.  2.) 
VI.  Vibrio  spermr^tozoides  Loffler.      x  1,000.      (Cop- 
ied from  Loffler:  C.  B.,  VII.,  PI.  III.,  Fig.  7.) 
VII.  Spirochsetes  from  mucus  of  the  gums.      (Copied 

from  Loffler:  "Bakterien,"  PI.  I.,  Fig.  4.) 
VIII.  Recurrens  spirilla.  Human  blood,  smear  prepara- 
tion. (Copied  from  Fraenkel  and  Pfeiffer: 
"Atlas,"  No.  134.) 
IX.  Eecurrens  spirilla.  Human  blood,  spirilla  ar- 
ranged in  a  stellate  shape.  (Copied  from  M. 
J.  Soudakewitsch :  Annates  de  V  Inst.  Pasteur, 
Vol.  v.,  1891,  p.  514,  PI.  14,  Fig.  1.) 


58 


Tab.  50. 


#. 


1 


11. 


□  CO] 


ffl. 


iv; 


VTl 


VI. 


*  t  € 


vm 


DC. 


THE  PROPERTY  OF 

llmeDiaBD  Medical  Coliegeef  tie  ra< 


Tab.  51. 


Explanation  of  Plate  59. 

Leptothrix  epidermidis.     Biz. 

I.  Gelatin  stick  culture,  two  days  at  22°. 
II.  Agar  streak  culture,  two  days  at  22°. 

III.  Agar  stick  culture,  two  days  at  22°.     Puncture 

canal. 

IV.  Agar  stick  culture,  two  days  at  22°.     Surface. 
V.  Agar  plate,  two  days  at  22°.     Natural  size. 

VI.  Agar   plate,   two   days  at  22°.      x90.     Part  of 
superficial  colony. 
VII.  Agar  plate,  two  days  at  22°.      x  90.    Deep  colony. 
VIII.  Gelatin  plate,  two  days  u.t  22°.     Natural  size. 
IX.   Gelatin  _ plate,   one  day  at  22°.     e,   Superficial; 
if  deep  colony. 
X.  Potato  culture,  three  days  at  22°.     Natural  size. 
XI.  Microscopical   preparation.     Pure    agar    culture, 
two  days  at  22°.      x  1,000.     Fuchsin  stain. 
XII.  Microscopical   preparation.     Bouillon   culture  in 
hanging  drop,  two  days  at  22°.      x  1,000. 


59 


Explanation  of  Plate  60. 

OospoRA  FARciNicA  (Noccaid)  Sauv.  and  Rad. 

I.  Agar  streak  culture,  eight  days  at  22°. 
II.  Gelatin  stick  culture,  twelve  days  at  22°. 

III.  Agar  stick  cultutre,  eight  days  at  22°.     Puncture 

canal. 

IV.  Agar  stick  culture,  eight  days  at  22°.     Surface. 
V.  Gelatin  plate,  ten  days  at  22°.     Natural  size. 

VI.  Gelatin  plate,  ten  days  at  22°.     x60.     Superficial 
(e)  and  doep-seated  (1)  colonies. 
VII.  Agar  plate,  six  days  at  22°.     Natural  size. 
VIII.  Agar  plate,  aight  days  at  22°.     Upper  colony  sur 
perficial ;  lower  one  deep. 
IX.  Potato  culture,  soven  days  at  22°.     Natural  size. 
X.  Microscopical   preparation.      Bouillon   pure  cul- 
ture, two  days.     x800.     Stained  with  fuchsin. 


60 


Tab.  52. 


^^^^^^^^^^^ 

r* 

' 

■^ 

/ 

e 

Tab.  61. 


Explanation  of  Plate  61. 

OospoRA  CHROMOGENES.     Lehm.  and  Neum. 

(Cladothrix  dichotoma  Autorum  non  Colin. ) 

I.  Gelatin  stick  culture,  six  days  at  22°. 
II.  Agar  streak  culture,  six  days  at  22°. 

III.  Agar  stick  culture,  six  days  at  22°.     Puncture 

canal. 

IV.  Agar  stick  culture,  six  days  at  22°.     Surface. 

V.  Gelatin  plate,  eight  days  at  22°.     Natural  size. 
View  upon  a  white  background. 
VI.  Gelatin  plate,  eight  days  at  22°.     Natural  size. 

View  upon  a  black  background. 
VII.  Gelatin  plate,  eight  days  at  22°.      x  60.     Part  of 
a  superficial  colony. 
VIII.  Agar  plate,  four  days  at  22°.     x60.     Superficial 
and  deep  colony. 
IX.  Potato  culture,  three  days  at  22°.     Natural  size. 
X.  Microscopical  preparation.      Bouillon   pure   cul- 
ture, three  days  at  22°.    x  1, 000.     Stained  with 
fuchsin. 


61 


Explanation  of  Plate  62. 

OospoRA  BO  VIS.     (Harz.)  Sauv.  and  Ead. 

(Actinomyces.) 

I.  Agar  streak  culture,  six  days  at  37°. 
II.  Agar  streak  culture,  thirty  days  at  37°. 
III.  Gelatin  stick  culture,  fourteen  days  at  22°. 
IV.  Gelatin  plate,  six  days  at  22°.     Natural  size. 
V.  Agar  plate,  six  days  at  37°.     Natural  size. 
VI.  Agar  plate,  six  days  at  37°.      x60.     Superficial 

and  deep  colony. 
VII.  Gelatin  plate,  six  days  at  22°.     xOO.     Superficial 
and  deep  colony. 
VIII.  Potato  culture,  ten  days  at  37°.     Natural  size. 
IX.  Microscopical  preparation.    Bouillon  pure  culture, 
three  days  at  37°.      x  1,000.     Fuchsin  stain. 


Tab    62. 


VI 


vn. 


IX. 


Tab    63. 


Explanation  of  Plate  63. 

Mycobacterium   lepr^    (Arm.    Hansen)    Lehm.    and 
Neum. 

Bacterium  influenza,  E..  Pfeiffer. 

Bacterium  pestis,  Lehm.  and  Neum. 

I.  Mycobacterium  leprae.  Giant  cell  from  leprous 
ulcer  of  epiglottis.  xljOOO.  Stained  by  Kus- 
elP  s  method.  (Copied  from  Seif ert  and  Kahn  : 
"Atlas  d.  Histopath.  d.  Nase,"  1875,  PI.  38, 
Fig.  75  b.) 

II.  Mycobacterium  leprae.  Transverse  section  of 
blood-vessel  in  a  leprous  testicle;  bacilli  in 
endothelium  and  in  a  white  blood  globule. 
Stained  according  to  Gram  and  with  Bismarck 
brown,  eosin,  bergamot  oil.      x  1,000. 

III.  Mycobacterium  leprae.  Longitudinal  section  of 
ulnar  nerve.  Staining  as  above.  (Copied 
from  Lie :  "  Path.  Anatomic  d.  Lepra, "  Arch, 
f.  Dermatol,  und  Syi^h.,  Vol.  XXIX.,  1895, 
PI.  VI.) 

IV.  Streptobacilli  in  soft  chancre.  Section  of  an  un- 
treated chancre,  twelve  days  old.  Stained  by 
Unna's  method.  (Copied  from  Petersen :  "  Ue- 
ber  Bacillenfund  bei  Ulcus  molle,"  C.  B., 
XIIL,  PI.  4.) 
V.  Bacterium  influenzae.  Smear  preparation  from 
the  nasal  secretion,  x  1,000.  Stained  with 
fuchsin. 

VI.  Bacterium  pestis.  Smear  preparation  from  lym- 
phatic gland  of  a  rat  which  died  suddenly. 
X  1,000.  (Copied  from  Yersin,  semi-schematic 
on  account  of  imperfect  photogram,  Annates  de 
VInstitut  Pasteur,  1894,  PI.  XII.,  Vol.  8, 
Fig.  2.) 
VII.  Bacterium  pestis.  Bouillon  pure  culture.  (Cop- 
ied, as  r.bove.  Fig.  3.)      x  1,000 

63 


A.  Introduction  to  the  Morphology  of 
Bacteria. 

By  the  term  bacteria  (schizomycetes  of  Naegeli)  is 
meant  a  very  large  group  of  the  lowest  vegetable  or- 
ganisms, which  are  morphologically  very  simple  and 
uniform,  but  biologically  are  extremely  differentiated. 
They  are  related  to  the  lowest  algae  (pliycochroma- 
cea)  and  the  lowest  fungi  by  so  many  intermediate 
forms  that  a  strict  separation  by  a  rigid  definition 
appears  difficult.  Various  bacteria  also  exhibit  great 
similarity*  to  the  simplest  flagellates,  which  are  usu- 
ally regarded  as  animals. 

A  definition  is  rendered  more  difficult  by  the  fact 
that  botanical  investigations  of  bacteria  are  compara- 
tively rare,  and  that  we  still  possess  very  imperfect 
knowledge  concerning  various  details  in  the  struc- 
ture of  bacteria  (ramifications,  separately  stained 
parts  t). 

*  Vide  Biltschli  in  Bronn's  "  Klassen  des  Tierreiches,  "  vol.  i., 
part  ii.,  Mastigophora. 

f  It  is  to  be  noted,  moreover,  that  according  to  Brefeld's  my- 
cological  investigations  (vol.  viii.,  p.  274),  forms  develop  dur- 
ing the  process  of  development  of  higher  fungi  which  possess  a 
striking  resemblance  to  bacteria  during  many  successive  genera- 
tions. We  must  therefore  concede  the  possibility  that  among 
the  varieties  of  bacteria  a  number  do  not  deserve  the  term 
"  species, "  but  belong  to  the  category  of  other  fungi. 
5 


66 


ATLAS   OF   BACTERIOLOGY. 


The  following  definition  will  suffice,  perhaps,  for 
the  practical  requirements  of  bacteriology  : 

Small  unbranched  *  cells  (almost  f)  always  free  from 
chlorophyll,  with  a  thickness  which  is  hardly  ever 
more  than  2  p.,  and  extremely  rarely  3-5  jj-  ;  they  have 
a  globular,  rod,  thread,  or  screw  shape,  without  any 


a     ^6/ 


\c/ 


\d  y 


m 


••%#^**^ 


i 


Fig.  1. — The  Forms  of  Bacteria  according  to  Buchner. 


other  organs  than  flagella  which  serve  for  movement. 
Yegetative   proliferation    takes   place  by   transverse 

*  Concerning  our  knowledge  of  branched  bacteria,  mde  pp.  67 
and  68. 

f  Practically  important  bacteria  containing  chlorophyll  are  un- 
known. But  J.  Frenzel's  green  tadpole  bacillus  must  probably 
be  classed  among  the  schizomycetes.  The  position  of  Dangeard's 
eubacillus  multisporus  among  the  bacteria  seems  more  doubtful. 
L.  Klein  described  colorless  forms  with  bluish-green  spores. 


MORPHOLOGY  OF  BACTERIA.  67 

division,  very  rarely  by  longitudinal  division.  One 
series  of  forms  develop  endogenous,  round,  permanent 
spores;  in  others  conidia-like  constrictions  (arthro- 
spores)  have  been  observed  or  claimed.  Other  modes 
of  proliferation  are  unknown  at  the  present  time. 

So  far  as  we  know,  the  schizomycetes  occur  only  in 
the  forms  here  delineated,  and  which  were  first  com- 
pletely named  by  H.  Buchner. 

Forms  of  Solitary  Growth. 

Spherical  form  (a),  not  coccus. 

Oval  form  (6),  the  long  diameter  at  the  most  twice 
as  large  as  the  transverse  diameter. 

Short  rods  (c),  the  long  diameter  is  two  to  four 
times  the  transverse  diameter. 

Long  rods  (d) ,  the  long  diameter  is  four  to  eight 
times  the  transverse  diameter. 

Filamentous  shape  (e). 

Half-screw  or  comma  (/),  a  very  short  section  of  a 
screw,  at  the  most  a  half  winding. 

Short  screw  (g) ,  a  short  screw  winding. 

Long  screw  or  spiral  form  {h).  All  screw  forms 
may  occur  either  with  steep  or  flat  threads. 

Spindle  form  {i). 

Oval  rods  (/?)  are  distinguished  from  the  spindle 
form  by  the  lesser  attenuation  of  the  extremities; 
from  the  oval  form  by  their  greater  length  (two  to 
four  times  the  transverse  diameter) . 

Club  form  {l). 

Growth  in  Groups. 

Double  spheres  (m),  with  the  separation  merely 
indicated;  roll  shape  or  biscuit  shape  (n). 


68  ATLAS   OF   BACTERIOLOGY. 

Spherical  series  (o),  up  to  eight  spheres,  with  the 
separation  merely  indicated;  torula  shape  (p). 

Spherical  threads  (q),  or,  if  curved,  rosary  shape 
(s) ;  with  the  separation  merely  indicated ;  filaments 
free  from  torula  (r) . 

Grape  shape  (^).  Double  rods  (u).  Filaments  of 
links  (v). 

Tetrads  (w),  a  combination  upon  one  plane  of  four, 
eight,  sixteen,  or  more  cells. 

Dice  shape  (x),  a  combination  of  eight,  thirty -two, 
etc.,  cells. 

The  formation  of  branches  (dichotomy),  i.e.,  the 
production  of  a  lateral  sprout  in  bacteria,  was  un- 
known until  recently,  and  is  at  all  events  rare.  It  has 
been  demonstrated  positively  in  the  tubercle,  diphthe- 
ria, and  glanders  bacilli  (vide  Plate  48,  Fig.  YIII.), 
so  that  for  the  present  these  varieties  occupy  a  posi- 
tion between  the  bacteriaceae  proper  and  the  hy  phomy- 
cetes  or  filamentous  fungi. 

A  different  interpretation  attaches  to  pseudodichot- 
omy,  which,  according  to  Babes  (Z.  H.,  XX.,  412), 
occurs  not  very  rarely  in  the  most  typical  bacteria. 
Either  the  lower  link  of  a  filament  grows  past  the 
upper  one  and  to  one  side,  or,  in  a  coccus  series,  a 
division  of  a  coccus  parallel  to  the  direction  of  the 
filament  suddenly  initiates  the  beginning  of  a  sec- 
ond filament. 

Much  has  been  written  recently  concerning  the 
structure  of  the  bacterium  cell.  We  must  confine  our- 
selves to  a  mere  abstract. 

According  to  Biitschli  ("Ueber  den  Bau  der  Bak- 
terien,"  etc.,  Heidelberg,  Winter,  1890)  (Fig.  3),  the 
bacterium  cell  consists  of  a  membrane;   a  layer  of 


MORPHOLOGY   OF   BACTERIA. 


69 


plasma,  wliich  takes  haematoxylin  stain  with  diffi- 
culty, is  often  very  thin,  and  indeed  often  present 
only  at  the  extremities;    and  a  large    central  body 


B  1  P 


r/ 


/ 


^ 

0 

CS 

cs 

.0 

©, 

0 

0 

Q 

Ck 

0 

r\     _. 

^ 
% 

0 

0 

88 

80^ 

8 

8 

9 

8 

0 

/-* 

0 

0 

c/ 

8 

0 

O 

a  6 

Fig.  8.— Pseudodichotomy.    a.  In  bacilli;  6,  in  streptococci. 


(nucleus),  which  stains  better  with  hsematoxylin. 
The  latter  shows  a  distinct,  the  former  not  always  a 
distinct  honey-combed  structure.  Among  the  meshes 
of  the  comb,  which  stain  blue  with  hsematoxylin,  are 
situated  in  the  central  body  numerous  granules  which 
are  stained  red  by  haematoxylin. 
At  an  earlier  period  Schottelius  (C.  B.,  IV.,  705) 


Fig.  3.— Chromatium  Okenii 
Ehrbg.    (After  Butschli.) 


Fig.  4.— Bacillus  oxalaticus 
Migula.    (After  Migula.) 


expressed  a  similar  opinion  of  the  structure  of  bacte- 
ria. According  to  him  the  bacillus  anthracis  consists 
of  a  narrow  nuclear  filament,  which  stains  a  blackish- 
red  with  a  very  dilute  watery  solution  of  fuchsin,  and 


70  ATLAS   OF   BACTERIOLOGY. 

a  protoplasmic  body,  which  does  not  stain  so  readily. 
These  two  structures  together  constitute  the  bacillus 
as  ordinarily  conceived;  they  are  surrounded  by  a 
membrane  which  stains  with  difficulty  {vide  page  72). 
According  to  Alfred  Fischer  *  (Fig.  4),  the  condi- 
tions are  very  simple  and  essentially  different  from 
those  just  described.  The  bacterium  consists  of  a 
cell  membrane,  a  protoplasmic  tube,  and  a  central 
fluid;   nothing  is  yet  known  concerning  a  nucleus. 


Fio.  4  a.— Plasmolysis,  according  to  A.   Fischer,    a,  Spirillum   undula; 
6,  bacterium  Solmsii ;  c,  vibrio  cholerae. 

In  solutions  of  salts  (sodium  chloride,  potassium 
nitrate,  etc.) — and  the  more  rapidly  the  more  con- 
centrated the  solution — the  abstraction  of  water  pro- 
duces "plasmolysis,"  i.e.,  a  retraction  of  the  proto- 
plasmic tube  with  partial  detachment  from  the  cell 
wall,  t  This  explains  numerous  bright  vacuoles  which 
develop  in  many  bacilli  on  making  an  ordinary  cover- 
glass  preparation,  and  which  were  formerly  often 
regarded  as  spores. 

At  the  same  time  and  independently  of  A.  Fischer, 
*  "  Untersuchungen  iiber  Bakterien,  "  1894,  Berlin.      Reprint 
from  the  Jahrb.  f.  wiss.  Botauik,  xxvii.,  vol.  1, 

f  Desiccation  on  the  cover-glass  often  suffices  to  produce  pic- 
tures of  plasmolysis. 


MORPHOLOGY   OF   BACTERIA.  71 

Migula  arrived  at  the  same  conclusions  from  a  study 
of  the  very  large  bacillus  oxalaticus,  a  sporulating 
variety  related  to  the  hay  bacillus.  He  emphasizes 
particularly  the  fact  that  he  has  never  succeeded  in 
staining  the  "central  body"  darker  than  the  proto- 
plasm. In  the  protoplasmic  tube  which  has  been 
squeezed  out  of  the  cell  membrane,  the  central  space 
for  the  fluid  can  be  made  esi)ecially  distinct  by  the 
fact  that  in  media  which  abstract  water  it  becomes 
smaller ;  in  water  it  becomes  larger. 

In  very  many  varieties  the  interior  of  the  bacterium 
cells  is  found,  after  suitable  staining,  to  contain  pecu- 
liar granules.  Babes,  their  discoverer,  applied  to 
them  the  non-committal  term  metachromatic  gran- 
ules (i.e.y  staining  differently  than  the  body  of 
the  bacterium),  while  Ernst,  their  first  thorough 
investigator,  terms  them  nuclei  or  sporogenous 
granules. 

For  the  literature,  which  is  rich  in  controversy, 
I  refer  to  Babes  (Z.  H.,  XX.,  412),  and  here  will 
merely  give  the  very  plausible  and  clear  views  of  K. 
Bunge,  the  most  recent  student  of  the  subject.  Bunge 
{Fort.  d.  Med.,  XIII.,  1895)  distinguishes: 

1.  Ernst's  granules.  They  are  stained  by  warm 
Loffler's  methyl  blue,  and  are  differentiated  black- 
ish blue  by  a  solution  of  Bismarck  brown^  but  they 
disappear  on  boiling.  These  granules  are  entirely 
absent  in  some  sporulating  varieties  (anthrax,  mega- 
therium) ;  in  others  it  can  be  proven  that  they  have 
no  relation  to  spores — hence  they  are  cell  granules  of 
unknown  rank. 

2.  Sporule  preliminary  stages  (Bunge 's  granules). 
Small  granules,   the  majority  of  which  are  usually 


72 


ATLAS   OF   BACTERIOLOGY. 


fouDd  in  tlie  sporulating  cells ;  they  are  not  stained 
by  Ernst's  method,  but  stain  in  boiling  Loffler's 
solution.  After  preliminary  treatment  of  the  dried 
preparation  with  chromic  acid,  sodium  hyperoxide, 
or  hydrogen  hyperoxide,  they  are  best  shown  by  the 


Bacterium  pneumoniae      Bacillus  anthracis 
(Friediander).  (Cohn). 

Fig.  5.— Formation  of  a  Capsule.     (Schematic.) 


streptococcus   lanceolatus 
(Gamal.). 


ordinary  spore  staining  (vide  Technical  Appendix). 
The  mature  spore  is  produced  by  the  union  of  several 
small  preliminary  stages. 

Bunge  explains  the  controversies  by  the  frequent 
confusion  of  the  two  different  varieties  of  granules. 

Concerning  the  cell  membrane,  it  is  to  be  noted  that 
often  it  is  not  sharply  defined  on  the  outside  and  ap- 
pears somewhat  swollen.  In  some  varieties  of  bacte- 
ria ("  capsule  bacteria"  of  writers)  the  thickening  of 
the  membrane  or  of  the  outer  layer  of  the  membrane 
is  so  great  that  the  bacterium  appears  to  be  surrounded 
by  a  veritable  mucous  envelope  or  capsule,  which  is 
characterized  by  its  slight  response  to  staining  with 
aniline  colors.  It  is  an  interesting  fact  that  these  bac- 
teria form  capsules  only  when  they  grow  in  the  ani- 
mal body  or  upon  special  nutrient  media,  such  as  fluid 
blood   serum,    bronchial   mucus,    and,    according    to 


MORPHOLOGY   OF   BACTERIA.  73 

Paulsen,  milk.*     The  capsules  do  not  form  when  the 
cultures  are  made  on  gelatin,  agar,  and  potatoes. 

Peculiar  unilateral  thickenings  or  swellings  of  the 
membrane  are  found  in  bacterium  pediculatum,  which 
is  described  as  a  rare  cause  of  the 
"frog-spawn  disease"  of  sugar  fac- 
tories (Fig.  6). 

In  the  spherical  forms  the  outer     fiq.  6.  -b  a  c  t  e  r  i  u  m 
surface  of  the  bacteria  is'  almost  al-        pediculatum.  (After 

, ,       .      ,  1         1        ,         -I     ',  '  Koch  and  Hosfius. ) 

ways  smooth ;  m  the  short  rods  it  is 
often  smooth  and  without  appendages,  but  the  larger 
rod  and  screw  forms  are  usually  provided  with  single 
or  numerous  thin  flagella.  These  are  sometimes  dis- 
tributed over  the  entire  body  of  the  bacterium,  some- 
times they  form  a  bundle  at  one  pole,  sometimes  there 
is  only  a  single  polar  flagellum.  Shortly  before  divi- 
sion bacteria  with  polar  position  of  the  flagella  show 
one  flagellum  or  a  bundle  of  flagella  at  each  pole. 
As  A.  Fischer  clearly  proved,  the  flagella  are  not 
structures  similar  to  the  retractile  and  extensible 
pseudopodia,  but  are  true  hair-like  formations  which 
develop  from  outgrowth.  In  order  to  color  the  fla- 
gella it  is  necessary  to  treat  the  bacteria  with  unusu- 
ally powerful  staining  reagents.      Then    the    mem- 

*It  is  not  certain  that  pronounced  capsule  formation  alwa^'S 
takes  place  in  these  nutrient  fluids.  Recently  various  authors 
have  called  attention  to  the  fact  that  capsule-like  formations  are 
observed  extensively  among  bacteria.  Johne  describes  a  method 
by  which  they  are  easily  made  visible  in  anthrax,  and  distinct 
capsules  are  also  seen  in  this  way  in  bacillus  megatherium,  bacillus 
oxalaticus,  etc.  Bab6s  has  depicted  capsules  in  streptococcus 
pyogenes,  and  we  have  occasionally  seen  similar  appearances  in 
many  bacteria.  Masses  of  bacteria  which  are  united  into  mucous 
clumps  by  swelling  of  the  capsules  are  called  "  zooglcea.  " 


74  ATLAS   OF   BACTERIOLOGY. 

brane,  which  usually  remains  colorless  with  ordinary- 
stains,  is  also  stained  and  the  bacteria  appear  to  be 
much  thicker.  Occasionally  broad  layers  of  the 
membrane  remain  unstained,  and  the  flagella  are  then 
situated  upon  a  narrow  annular  areola,  separated  from 
the  bacillus  by  a  colorless  zone  (Zettnow,  von  Stock- 


5E/ 


r\^ 


-4     ^ 


a  h  c 

Fig.  7.— Types  of  Flagella.  a,  Vibrio  oholerse,  a  flagellum  at  one  ex- 
tremity; 6,  bacterium  syncyaneum,  a  bundle  of  flagella  at  one  extrem- 
ity, rarely  on  the  side;  c,  bacterium  vulgare,  flagella  arranged  round 
about. 

lin,  A.  Fischer).  Unfortunately  many  of  the  meth- 
ods used  in  staining  lead  forthwith  to  exfoliation  and 
degeneration  of  the  flagella,  so  that  their  perfect  ex- 
hibition is  often  difficult.  The  above  figure  gives 
a  schematic  representation  of  the  three  modes  in 
which  bacteria  are  provided  with  flagella. 

In  the  cultures  of  bacteria  which  are  rich  in  flagella, 
Loffler  first  observed  the  occasional  production  of 
peculiar,  switch-like  bodies,  composed  of  flagella 
which  had  fallen  off  or  had  been  cast  off  and  were 
plaited  into  one  another  (vide  Plate  47,  Fig.  II.). 

The  power  to  produce  flagella  may  be  lost  entire- 
ly for  generations ;  whether  permanently  is  ^ill  un- 
known. Vide  Micrococcus  agilis,  sarcina  mobilis 
(Lehmann  and  Neumann) . 

Ordinary  vegetative  increase  of  bacteria  is  effected 


MORPHOLOGY   OF   BACTERIA.  75 

by  a  transverse  constriction  in  the  middle  of  the  bac- 
terial cell,  which  has  either  been  very  little  (spherical 
bacteria)  or  considerably  elongated.  As  a  rule,  the 
micro-organisms  separate  soon  after  fission,  but  the 
opposite  event  may  occur  in  all  groups  of  bacteria, 
so  that,  for  example,  chains  of  spheres  or  rods  de- 
velop. Under  certain  nutritive  conditions  the  bacte- 
riacese,  vibriones,  and  higher  bacteria  give  rise  to  the 
production  of  long  threads,  but  later  these  may  again 
be  resolved  into  links.  According  to  all  recent  inves- 
tigations, division  of  the  cell  starts  in  the  protoplas- 
mic layer  upon  the  wall,  the  central  "nucleus"  or 
"cavity"  is  divided  passively,  and  the  cell  membrane 
takes  part  secondarily.  This  is  evidently  opposed  to 
the  interpretation  of  the  central  body  as  a  nucleus, 
because  division  of  the  nucleus  always  precedes  divi- 
sion of  the  cell. 

Longitudinal  growth  with  transverse  fission  is  the 
rule  for  the  mass  of  bacteria,  but  in  certain  forms — 
for  example,  sarcina — there  is  a  regular  alternation  of 
the  fission  in  the  three  principal  planes.  At  least 
occasional  division  along  two  planes  at  right  angles 
to  one  another  has  been  observed  in  very  different 
bacteria — for  example,  in  streptococci — and  thus  cells 
in  four  parts  may  develop,  with  bifurcation  of  the 
chain  {vide  Fig.  2). 

Longitudinal  division  of  rod  forms  has  been  ob- 
served rarely  but  undoubtedly  (Babes:  Z.  H.,  XX.). 
Metschnikoff  observed  stellate  division  in  a  sporu- 
lating  organism  known  as  "Pasteuria,"  but  this  can 
hardly  be  classed  among  the  bacteria  in  the  narrower 
sense. 

Ordinary  vegetative  proliferation  must  be  distin- 


76  ATLAS   OF   BACTERIOLOGY. 

guished  from  that  due  to  the  formation  of  spores. 
We  are  acquainted  to-day  with:  (1)  Endospores, 
strongly  refracting  oval  or  round  bodies  developing  in 
the  interior  of  the  cell,  and  which  as  a  rule  possess 
considerable  resistance  to  injurious  influences  (heat, 
dryness,  chemicals) ;  and  (2)  arthrospores  (De  Bary, 

Hiippe),  i.e.y  sprout-like 
constriction  of  one  end  of 
the  cell.  These  spores 
(Fig.  8)  are  also  said  to 
Fig.  8.  —  Arthrospores  of  Vibrio  exhibit    increased    resist- 

choleraB,  according  to  Huppe.         ^^^^^      ^^^    ^^    ^-^^    ^^^^^^ 

investigations  have  furnished  no  absolutely  positive 
proof  of  the  formation  of  arthrospores  which  ex- 
hibit increased  resistance,  the  difficult  question  of 
arthrospores,  important  as  it  is,  must  be  regarded 
as  still  open. 

In  the  following  pages  the  term  spores  refers  only 
to  endogenous  permanent  forms. 

In  the  different  varieties  the  development  of  the 
endospores  runs  a  similar  but  not  identical  course. 
In  examining  any  definite  variety  for  the  development 
of  spores,  we  resort,  as  a  rule,  to  agar  streak  or  po- 
tato cultures,  which  are  kept  at  a  temperature  best 
adapted  to  the  variety  in  question.  At  the  end  of 
twelve,  eighteen,  twenty-four,  thirty,  thirty-six  hours, 
we  examine  small  tests  of  the  streak  culture,  first  un- 
stained in  water  and  with  a  narrow  angle  of  aperture. 
If  it  is  thought  that  round  or  oval,  strongly  refracting 
spores  have  been  found,  the  spores  are  stained  ac- 
cording to  Neisser's  or  Hauser's  method  (vide  Tech- 
nical Appendix) .  For  the  careful  study  of  the  devel- 
opment of  spores,  it  is  best  to  place  a  few  bacilli  in  a 


MORPHOLOGY   OF   BACTERIA.  77 

hanging  drop  of  gelatin  or  agar,  and  (if  necessary, 
with  the  aid  of  warming  apparatus  or  in  a  well-heated 
room)  to  observe  and  draw  definite  individual  cells 
uninterruptedly. 

Motile  varieties  always  come  to  a  standstill  (ac- 
cording to  A.  Fischer)  before  the  formation  of  spores, 
but  they  do  not  cast  off  their  flagella.  Certain  varie- 
ties first  grow  into  long  filaments,  which  at  the  be- 
ginning are  unsegmented.  This  variety  includes  the 
bacillus  anthracis,  whose  sporulation  will  be  selected 
as  an  example  {vide  Plate  40,  Figs.  VI.  and  III.). 

The  previously  homogeneous  bacteria  first  exhibit 
a  delicate,  cloudy  opacity ;  then,  according  to  Bunge, 
the  very  finest  granules  are  replaced  by  a  smaller 
number  of  somewhat  coarser  granules,  which  coalesce 
until  small,  rounded  spores  are  situated  at  regular 
intervals  (Plate  40,  Fig.  VI.),  and  are  converted  grad- 
ually into  the  oval,  strongly  refracting,  mature  spores 
(Plate  40,  Fig.  III.). 

When  sporulation  is  complete,  we  find  in  the  bac- 
terial filament  a  delicate  septum  between  two  spores 
(Plate  40,  Fig.  IV.).  Not  all  segments  which  have 
begun  the  development  of  spores  by  the  formation  of 
spherical  preliminary  stages  mature  these  spores. 
Indeed,  certain  varieties,  as  the  result  of  various  con- 
ditions of  culture,  gradually  suffer  a  permanent  loss 
of  the  power  of  producing  mature  spores  and  form 
only  preliminary  stages,  which  are  physiologically 
valueless  (Eoux,  K.  B.  Lehmann). 

According  to  Lud.  Klein  (C.  B.,  VII.,  440),  spor- 
ulation is  entirely  different  in  five  usually  motile,  an- 
aerobic forms  of  bacilli  (bacillus  De  Baryanus,  Solm- 
sii,  Peromelia,  macrosporus,  limosus),  which  were 


78  ATLAS   OF   BACTERIOLOGY. 

discovered  and  studied  by  him  (but  unfortunately  not 
in  pure  cultures) .  In  these  the  process  ran  the  fol- 
lowing course :  Without  any  cessation  in  the  motion 
of  the  bacillus,  one  extremity  becomes  somewhat  en- 
larged and  acquires  a  slightly  greenish  tinge.     The 


^ 


^ 


/? 


<^ 


Fig.  9.— Types  of  Spores. 


entire  contents  of  the  distended  part  now  contract 
into  a  spore  of  bluish-green  color  and  striking  bril- 
liance. 

In  the  most  important  varieties  the  mature  spores 
appear  as  follows  (Fig.  9) : 

1.  The  spore  lies  in  the  interior  of  a  non-dis- 
tended, short  bacterium  cell  (a). 

2.  The  spore  lies  in  the  interior  of  a  non-distended, 
short  bacterium  cell,  which  forms  merely  a  link  of  a 
long  filament  (b). 

3.  The  spore  lies  in  the  interior  of  a  bacterium  cell, 
which  has  been  distended  in  the  middle  and  has  be- 
come spindle  shaped  (d). 

4.  The  spore  lies  at  the  extremity  of  a  non-distended 
short  bacterium  cell,  apparently  projecting  far  beyond 
it  (c). 

The  germination  of  spores  has  been  little  studied. 
They  are  generally  set  free  before  germination  by  rup- 
ture of  the  filament.     An  outgrowth  of  the  spores  in 


MORPHOLOGY   OF   BACTERIA. 


79 


the  bacillus  at  right  angles  to  the  direction  of  the  fila- 
ment is  rarely  observed  {vide  Sorokin,  C.  B.,  I.,  465). 

The  following  cut  shows  the  germination  of  a  few 
closely  allied  varieties  which  were  studied  by  L. 
Klein. 

The  examination  is  made  in  a  hanging  drop  of  gel- 
atin or  agar.     This  may  furnish  very  valuable  mate- 


Z     3 


3^5 


°os/ 


Fig,  10.— Development  of  Spores,  according  to  L.  Klein,  a,  Bacillus 
leptosporus  L.  Klein;  1-3,  the  enlarging  spore;  4,  the  spore  is  con- 
verted into  the  bacillus  without  any  sharp  demarcation ;  5-10,  further 
growth ;  11-16,  development  of  the  spores.  6,  Bacillus  sessilis  L,  Klein ; 
1-4,  the  spore  swells;  5,  the  spore  sends  out  a  little  rod  at  one  pole, 
and  remains  behind  as  an  empty  envelope;  6-8,  further  growth;  9-13, 
development  of  the  spores. 

rial  in  differential  diagnosis,  as  it  seems  to  differ 
greatly  in  details. 

1.  Bacillus  anthracis.  The  spore  swells,  its  refrac- 
tive power  diminishes,  its  sharply  defined  membrane 
becomes  indistinct,  and  without  any  sharp  demarca- 
tion the  spore  becomes  a  young  bacterium  cell,  which 
grows  further  and  divides  again. 

A  similar  condition  obtains  in  the  bacillus  lepto- 
sporus Klein,  described  by  L.  Klein  (C.  B.,  VI.,  377), 
which  is  characterized  by  narrow,  almost  quadrangu- 
lar spores  (Fig.  10,  a). 


80  ATLAS   OF    BACTERIOLOGY. 

2.  Bacillus  subtilis  Cohn.  The  membrane  of  the 
growing  spore  bursts  at  the  equator,  the  firm-walled 
membrane  of  the  spore  adheres  not  infrequently  to 
the  emerging  young  rod,  even  after  it  has  grown  into 
a  long  filament. 

3.  Bacillus  sessilis  Klein.  The  spore  enlarges  to  a 
marked  degree,  then  ruptures  at  one  pole,  and  from 
the  envelope  of  the  spore  grows  a  motionless  filament, 
to  which  the  yellowish-green,  contracted  membrane 
of  the  spore  remains  adherent  for  a  very  long  time 
(Fig.  10,  h). 

In  old  cultures  of  bacteria  we  almost  always  find 
dead,  often  very  queerly  shaped  bacterial  cells  (invo- 
lution, degeneration  forms),  which  are  shown  in  Plate 
40,  Fig.  Y.,  and  Plate  53,  Fig.  YI.  These  swollen, 
bent,  often  unrecognizable  forms  stain  poorly  with  the 
ordinary  reagents.  The  beginner  will  often  regard  in- 
volution forms  as  the  result  of  fouling ;  the  resort  to 
plate  cultures  will  soon  show  whether  we  have  to  deal 
with  one  or  more  forms  of  bacteria. 


B.   The   Chemical   Composition   of  Bacteria. 

The  body  of  bacteria  consists  in  great  imrt  of  water, 
salts,  and  albuminoids ;  *  extractive  matters  which 
are  soluble  in  alcohol,  and  other  bodies  (triolein, 
tripalmitin,  tristearin,  lecithin,  cholesterin)  which 
are  soluble  in  ether,  are  present  in  smaller  amounts. 

*  Albumin  and  salts  may  amount  to  ninety -eight  per  cent  of 
the  dried  bacteria  (cholera  vibrio),  and  on  the  other  hand  as 
much  as  twelve  per  cent  of  carbohydrates  may  be  present  in  the 
membranes.  Hellmich  found  a  globulin  in  the  bacterial  albu- 
min (Arch.  f.  exp.  Path.  u.  Pharm.,  xxvi.,  345). 


THE   CHEMICAL   COMPOSITION   OF   BACTERIA.  81 

In  no  variety  of  bacteria  could  E.  Cramer  discover 
grape  sugar;  some  varieties  (bacillus  butyricus,  lep- 
tothrix  forms)  contain  starch-like  masses  which  are 
stained  blue  b}^  iodine.  True  cellulose  was  discovered 
by  Drey  fuss  in  bacillus  subtilis  and  in  an  organism 
closely  related  to  bacterium  coli,  and  the  bacillus  tuber- 
culosis also  forms  cellulose  in  the  animal  body.  But 
no  cellulose  could  be  obtained  from  cultures  of  bacil- 
lus tuberculosis  and  a  "capsule  bacillus  from  water," 
closely  related  to  bacillus  pneumoniae  Friedlander, 
while  they  contained  a  large  amount  of  a  gelatinous 
carbohydrate  (CeHj^OJ,  which  is  closely  allied  to 
hemicellulose  (concerning  the  literature  vide  Nishi- 
mura:  A.  H.,  XYIII.,  318  and  XXI.,  52).  The  mu- 
cus of  leuconostoc  mesenterioides  was  shown  by 
Scheibler  {Ghem.  Centralhl.j  XI.,  181)  to  be  a  carbo- 
hydrate, C^Hj^Oa  dextro-rotatory.  Kramer  obtained 
a  similar  substance  from  the  membranes  of  bacillus 
viscosus  sacchari.  Nuclein  has  not  been  extracted, 
but  among  the  nuclein  bases  xanthin,  guauin,  and 
adenin  have  been  found  in  considerable  amounts. 
One  group  of  bacteria  deposits  suljjhur  granules, 
which  are  derived  from  sulphuretted  hydrogen  (beg- 
giatoa,  thiothrix) ;  another  variety,  which  is  classed 
among  bacteria  by  many  authors,  secretes  ferric  oxide 
into  its  membrane  from  ferruginous  waters  (cladothrix, 
crenothrix) . 

The  methodical  investigations  of  E.  Cramer  have 
shed  some  light  upon  the  quantitative  relations,  al- 
though accurate  statements  have  been  obtained  hith- 
erto only  concerning  bacterium  prodigiosum,  bacillus 
pneumoniae,  and  a  few  related  varieties,  and  a  series 
of  forms  of  vibrio  cholerae  {vide  E.  Cramer:  A.  H., 
6 


82  ATLAS   OF   BACTEEIOLOGY. 

XIII.,  70;  XVI.,  150;  and  XXII.,  167).  The  fol- 
lowing statements  and  figures  must  suffice  for  the 
limits  of  this  work. 

The  amount  of  water  in  a  culture  which  has  grown 
upon  a  solid  nutrient  medium,  and  likewise  the 
amount  of  ash,  depend  in  a  very  great  measure  upon 
the  composition  of  the  nutrient  medium. 

Eor  example,  bacterium  prodigiosum  contains, 
when  cultivated  on  potato,  21.49  per  cent  dry  sub- 
stance, 2.70  per  cent  ash  in  the  fresh  substance; 
when  cultivated  on  carrots,  12.58  per  cent  dry  sub- 
stance, 1.31  per  cent  ash  in  the  fresh  substance. 

Apart  from  the  concentration  of  the  nutrient  me- 
dium, higher  temperatures  and  youth  of  the  cultures 
increase  the  amount  of  dry  substance  and  ash. 

The  amount  of  dry  substance  of  bacteria  also  varies 
in  its  composition  in  the  same  variety,  under  the  in- 
fluence of  the  nutrient  medium. 

For  example,  the  bacterium  pneumoniae  Fried. ,  upon 
a  nutrient  medium  of  meat-infusion  agar,  contained : 

With  1  per         With  5  per 
cent  peptone,  cent  peptone. 

Albumin 71.7  79.8 

Ether  and  alcoholic  extract 10. 3  11. 28 

Ash 13.94  10.36 

« 

and  with  one  per  cent  peptone  and  five  per  cent  grape 
sugar : 

Per  cent. 

Albumin   63.6 

Ether  and  alcoholic  extract 22. 7 

Ash 7. 88 

It  is  evident  that  an  increase  in  the  amount  of  pep- 
tone in  the  nutrient  medium  causes   an  increased 


THE   CHEMICAL   COMPOSITION   OF    BACTERIA.  83 

amount  of  albumin  in  the  bacillus,  while  an  increased 
quantity  of  grape  sugar  diminishes  the  amount  of 
albumin. 

The  differences  are  much  greater  as  regards  the 
dry  substances  of  cholera  vibriones  when  cultivated 
upon  albuminous  soda  bouillon  and  upon  the  non- 
albuminous  Uschinsky  nutrient  medium.  Cramer 
found  (the  figures  represent  the  averages  from  experi- 
ments with  five  cholera  species)  that : 

Albumin.      Ash. 

Percent.  Percent. 

Cholera  vibriones  on  soda  bouillon  contained 65  31 

Cholera  vibriones  on  Uschinsky  solution  contained  .45  11 

In  the  latter  case  there  were  evidently  very  large 
amounts  of  non-nitrogenous  bodies,  which  may  be  re- 
garded in  part  as  hydrocarbons  (or  fats). 

A  very  important  point  in  classification — although 
more  in  a  critical  negative  sense — is  the  fact  discov- 
ered by  Cramer  that  closely  allied  varieties  which  ex- 
hibit analogous,  slightly  varying  composition  upon 
several  nutrient  media,  suddenly  act  differently  upon 
a  new  medium.  The  most  interesting  illustration  was 
the  conduct  of  five  cholera  varieties,  which  in  soda 
bouillon  produced  vibriones  of  almost  exactly  the 
same  constitution,  while  they  differed  greatly  in 
Uschinsky  solution: 

Soda  Bouillon. 

Albumin.  Ash.  Total. 

Cholera,  old 65.12  31.55  96.67 

Cholera,  Hamburg,  wmter  of  1893.  69.25  25.87  95.12 

Cholera,  Paris 62.25  32.80  95.05 

Cholera,  Shanghai 64. 25  33. 87  98. 12 

Cholera,     Hamburg,    autumn     of 

1893 63.94  29.81  93.75 


84  ATLAS   OF   BACTERIOLOGY. 


UscHiNSKY  Solution. 

Albumin.  Ash.  Total. 

Cholera,  old 48. 13  7. 14  55.27 

Cholera,  Hamburg,  winter  of  1892.  35.75  13.70  49.45 

Cholera,  Paris 65.63  9.37  70.00 

Cholera,  Shanghai 47. 50  1 1. 64  59. 14 

Cholera,    Hamburg,     autumn     of 

1893 34.37  14.74  49.11 

This  result  again  shows  how  dangerous  it  is  to  dis- 
tinguish two  varieties  by  relying  upon  a  single  chemi- 
cal or  biological  reaction.  Some  of  these  varieties 
need  merely  acquire  the  power  of  forming  thick  cell 
membranes  in  Uschinsky  solution  in  order  to  explain 
these  remarkable  differences.  How  easily,  for  exam- 
ple, could  a  writer  be  led,  from  these  figures,  to  re- 
gard the  bacilli  of  the  Paris  cholera  as  a  distinct  spe- 
cies, because  they  contain  almost  twice  the  amount 
of  albumin  in  Uschinsky  solution  as  those  of  the 
Hamburg  cholera. 

So  far  as  I  know  the  spores  of  bacteria  have  not 
been  closely  analyzed,  but  from  the  analogy  to  the 
spores  of  mould  fungi  we  may  expect  them  to  contain 
a  diminished  amount  of  water. 


C.  The  Vital  Conditions  of  Bacteria. 

1.    NUTRIENT  MEDIA. 

While  a  number  of  schizomycetes  have  been  found 
hitherto  only  in  the  human  or  animal  organism,  and 
therefore  appear  to  be  strict  parasites  (for  example, 
spirillum  Obermeieri),  the  majority  of  parasitic  vari- 
eties can  also  be  cultivated,  either  readily  (for  exam- 


THE   VITAL   COITDITIONS   OF   BACTERIA.  85 

pie,  bacterium  typhi)  or  with  difficulty  (for  example, 
micrococcus  gonorrhoeae)  in  artificial  nutrient  media. 
Among  the  inhabitants  of  the  inanimate  surroundings 
of  man,  the  so-called  saprophjtes^  the  majority  can  be 
easily  cultivated  in  the  same  artificial  media  as  para- 
sites; while  in  others,  for  example  certain  salivary 
and  water  bacteria,  such  cultivation  meets  with  in- 
surmountable obstacles. 

All  nutrient  media  must  be  rich  in  water,  and  the 
presence  of  salts  and  of  a  supply  of  carbon  and  nitro- 
gen is  also  indispensable.  The  majority  of  the  prac- 
tically important  and  all  the  pathological  varieties 
have  a  predilection  for  albuminous  and  feebly  alka- 
line nutrient  media. 

The  demands  of  the  bacteria  upon  the  composition 
of  the  nutrient  media  vary  extremely.  As  Mead  Bol- 
ton showed,  a  number  of  water  bacteria  (bacillus 
aquatilis  Fliigge  and  bacillus  erythrosporus  Fliigge) 
are  satisfied  with  water  which  has  been  distilled  twice 
in  glass  vessels.  Here  the  proliferation  of  the  bacte- 
ria must  have  taken  place  either  at  the  cost  of  traces 
of  impurities  or  of  the  ammonia  and  carbonic  acid  of 
the  atmosphere. 

In  water  which  contained  ammonium  carbonate  as 
the  sole  source  of  carbon  and  nitrogen,  and  was  ac- 
cordingly freei  from  all  organic  nutritive  material, 
Heraeus  observed  abundant  proliferation  of  a  variety 
of  fungus — that  is,  a  development  of  cell  substance 
from  the  simplest  material,  such  as  occurs  otherwise 
only  in  the  higher  plants  which  work  with  chlorophyll 
in  combination  with  sunlight.  Hiippe  and  particu- 
larly Winogradsky  have  shown  the  correctness  and 
importance  of  this  obsers^ation  as  the  result  of  care- 


86 


ATLAS  OF   BACTERIOLOGY. 


ful  studies.  The  energy  necessary  to  the  albumin 
synthesis  seems  to  be  gained  by  oxidation  of  ammo- 
nia into  nitric  acid. 

Very  few  practically  important  bacteria  exhibit 
such  simplicity,  but  very  many  can  dispense  at  least 
with  albumin  in  the  nutrient  and  thrive  in  solutions 
of  very  simple  composition.  Formerly  cultures  in 
such  fluids  were  employed  very  often,  and  recently 
Uschinsky  has  again  resorted  to  simple  nutrients. 
But  instead  of  Uschinsky 's  somewhat  complicated  so- 
lution : 


Water 1,000 

Glycerin 30-40 

Sodium  chloride 5-7 

Calcium  chloride 0.1 


Magnesium  sulphate  ., .  0.2-0.4 

Dikalium  phosphate  . . .  3-2. 5 

Ammonium  lacticum  . . .  6-7 

Sodium  asparaginicum  .  3-4 


we  may  choose  much  simpler  solutions ;  for  example, 
on  the  recommendation  of  Voges  and  C.  Fraenkel 
{Hyg.  Rundschau,  1894,  No.  17)  for  one  litre: 

Sodium  chloride 5  gm, 

Neutral  commercial  sodium  phosphate 2  gm. 

Ammonium  lactate 6  gm. 

Asparagin   4  gm. 

In  this  fluid  (although  it  contains  no  sulphur)  there 
grow: 

Very  Well. 
Bacillus  subtilis  and  mycoides. 

Bacterium    syncyaneum,    pyocyaneum,  coli,  acidi  lactici, 
pneumoniaj,  mallei,  vulgare.     All  vibriones. 

Feebly. 


Micrococcus    pyogenes    a 

aureus. 
Streptococcus  pyogenes. 


Bacterium  typhi. 
Bacillus  anthracis. 


THE  VITAL   CONDITIONS   OF   BACTERIA.  87 

Not  at  All. 
Bacillus  tetani  I  Bacterium  eryaipelatos  sunm. 

Bacterium  murisepticum.     I  Bacterium  cuniculicida. 

The  addition  of  the  other  substances  recommended 
by  Uschinsky  did  not  cause  vigorous  growth  of  other 
varieties  (such  as  diphtheria  and  tetanus),  while  on 
the  addition  of  three  to  four  per  cent  glycerin  the 
medium  becomes  very  serviceable  even  for  the  tubercle 
bacillus. 

Although  cultures  in  the  simple  nutrient  media  just 
described  possess  great  theoretical  interest,  they  are 
used  very  little  for  purposes  of  differential  diagnosis. 

Much  more  frequent  use  is  made  of  flesh-water  pep- 
tone gelatin,  flesh-water  peptone  agar,  bouillon  (with 
or  without  the  addition  of  grape  sugar  or  milk  sugar), 
glycerin  agar,  milk,  potato  discs. 

We  must  always  have  these  on  hand,  because  no 
differential  diagnosis  is  possible  without  them,  and 
no  variety  can  be  properly  described  which  has  not 
been  tested  in  regard  to  its  condition  in  all  these  nu- 
trient media  (with  the  exception  of  glycerin  agar). 

Less  use  is  made  of  the  following  nutrient  media : 
potato  water,  lamb  bouillon,  blood  serum  (fluid  and 
firm),  serum  agar,  agar  smeared  with  blood,  meat, 
pieces  of  bread,  mashed  potatoes,  rice,  boiled  or  raw 


2.    REACTION  OF  THE  NUTRIENT  MEDIA. 

As  has  been  remarked  above,  the  large  majority  of 
bacteria,  especially  the  pathogenic  forms,  have  a 
predilection  for  neutral  or  feebly  alkaline   nutrient 


88  ATLAS   OF   BACTERIOLOGY. 

media.  Formerly  it  was  recommended  that  a  solu- 
tion of  soda  should  be  added  gradually  to  the  nutri- 
ent medium  until  it  turns  red  litmus  paper  to  a  faint 
blue  color. 

Every  chemist  knows  that  there  is  no  sharply  de- 
fined final  reaction  for  the  titration  of  phosphatic  nu- 
trient media  with  litmus ;  furthermore,  that  different 
litmus  papers  influence  the  result,  and  that,  finally, 
titration  is  quite  impossible  by  gaslight.  For  this 
reason  W.  K.  Schultz,  in  1891,  recommended  phenol- 
phthalein  as  an  indicator  in  agar  tritration,  and 
advised  that  8-10  c.c.  less  of  normal  soda  lye  be 
added  to  a  litre  of  the  nutrient  medium  than  is 
necessary  for  complete  neutralization  with  this  indi- 
cator. In  this  way  a  medium  is  obtained  whose 
reaction  is  suitable  to  very  many  bacteria,  but  there 
are  some  which  require  complete  neutralization  (C. 
B.,  X.,  52). 

Without  having  noticed  this  suggestion  I  conceived 
the  same  idea,  in  1892,  during  my  investigations  on 
bread  acids.  Since  1894,  the  neutral  gelatin  (or 
agar)  used  in  my  laboratory  as  a  nutrient  medium 
has  been  treated  with  as  much  soda  lye  as  is  neces- 
sary to  slight  reddening  of  an  addition  of  phenol- 
phthalein.  All  the  plates  in  this  Atlas  have  been 
made  according  to  such  cultures,  after  experiments 
on  five  important  bacteria  had  shown  that  the  addi- 
tion of  alkalies  and  acids  to  this  neutral  medium  had 
not  improved  their  growth.  Since  then,  Mr.  Winkler, 
a  student  of  medicine,  has  systematically  tested  the 
power  of  growth  of  the  large  majority  of  bacteria 
described  in  our  Atlas.  This  has  been  done  upon  the 
following  nutrient  media : 


THE  VITAL   CONDITIONS   OF   BACTERIA.  89 

1.  Upon  agar  which,  after  the  use  of  phenol- 
phthalein,  was  neutralized  with  normal  soda. 

2.  On  "acid"  agar,  i.e.,  neutral  agar  which  has 
been  treated  with  10  c.c.  of  normal  sulphuric  acid  to 
1  litre. 

3.  On  three  varieties  of  alkaline  agar,  ^.e.,  on  neu- 
tral agar  which  has  received  10,  20,  and  30  c.c.  of 
normal  alkali  to  1  litre. 

The  results  laid  down  in  Table  I.  show,  in  brief, 
that  almost  all  bacteria  thrive  well  upon  three  of 
these  nutrient  media. 

At  all  events  the  medium  made  neutral  by  phenol- 
phthalein  may  be  recommended  unreservedly  as  a 
universal  nutrient;  the  virulence  of  the  varieties  ex- 
amined by  us  (anthrax,  bacterium  coli,  mouse  sep- 
ticaemia, chicken  cholera)  was  also  well  maintained 
thereon. 

This  reaction  possesses  the  advantage  that  it  is 
easily  prepared  and  represents  a  sharply  defined 
point,  viz.,  that  in  which  all  the  free  acids  and  the 
acid  salts  are  converted  into  neutral  salts  (mono- 
sodium  phosphate  into  disodium  phosphate) . 

If  acid  media  are  to  be  employed,  it  is  best  to  start 
with  one  which  has  been  neutralized  with  phenol- 
phthalein,  to  which  10,  20,  or  30  c.c.  of  normal  acid 
per  litre  may  be  added.  According  to  Winkler  the 
first  degree  of  acidity  is  well  tolerated  by  almost  all 
bacteria.  According  to  Schliiter's  statements  (C.  B., 
XI.,  589),  which  are  confirmed  by  recent  publications, 
many  tolerate  a  much  higher  degree  of  acidity ;  even 
as  much  at  100  c.c.  of  normal  acid  per  litre,  according 
to  experiments  made  in  our  laboratory. 

Apart  from  yeast  and  mould  fungi,  acid  nutrient 


90  ATLAS   OF   BACTERIOLOGY. 

media  should  always  be  employed  as  auxiliaries 
when  we  have  to  deal  with  the  isolation  of  a  bacteri- 
um from  an  acid  medium.  In  counting  the  germs  in 
the  air,  earth,  water,  milk,  etc.,  the  neutral  medium 
should  always  be  used. 


3.    INJURY  TO  BACTERIA  BY  CHEMICAL  SUB- 
STANCES. 

In  the  presence  of  an  excess  of  acids  or  alkalies  we 
have  just  recognized  a  factor  which  exerts  an  inhibi- 
tory influence  on  development,  and,  in  still  greater 
intensity  produces  death.  The  most  varied  chemi- 
cals act  in  a  similar  manner  after  a  certain  degree  of 
concentration.  The  most  effective  substances  are 
known  as  antiseptics  or  disinfectants. 

With  Hiippe,  we  usually  distinguish  the  following 
degrees  of  action : 

1.  The  growth  is  not  disturbed  but  the  pathogenic, 
zymogenic  functions  are  weakened — attenuation,  miti- 
gation. 

2.  The  organisms  can  no  longer  proliferate  but  are 
not  killed — asepsis,  kolysepsis. 

3.  The  vegetative  conditions  of  the  micro-organisms 
are  destroyed  but  not  the   permanent  forms — anti- 


4.  The  vegetative  and  spore  forms  are  killed — 
sterilization  or  disinfection. 

Inasmuch  as  the  test  of  the  resisting  power  to 
chemicals  plays  a  minor  part  for  diagnostic  purposes, 
this  section  will  be  treated  very  briefly. 

The  following  plan  should  be  adopted  in  order  to 
determine  the  minimum  concentration  of  the  chemical 


THE   VITAL   CONDITION'S   OF   BACTERIA.  91" 

poison  which  will  just  produce  asepsis,  i.e.,  inhibi- 
tion of  development. 

For  example,  a  ten-per-cent  solution  of  the  disin- 
fectant is  prepared,  and  1,  0.5,  0.3,  0.1  c.c,  etc.,  are 
added  to  10  c.c.  of  liquefied  gelatin.  The  tubes  then 
contain  1  per  cent,  0.5  per  cent,  0.^  per  cent,  0.1 
per  cent  of  the  disinfectant;  stick,  streak,  or  plate 
cultures  are  then  made  with  the  bacterium  which  is 
to  be  tested.  We  may  also  inoculate  with  material 
which  contains  only  spores  (material  which  has  been 
freed  from  all  bacilli  by  heating  for  half  an  hour  to 
70°  C.)  in  order  to  note  whether  these  spores  grow 
into  cultures. 

Behring  makes  this  test  in  the  following  practical 
form :  From  the  fluid,  infected  nutrient  medium  (for 
example,  serum)  which  is  to  be  tested  a  drop  is 
taken  before  the  addition  of  the  antiseptic,  and,  after 
being  placed  on  the  lower  surface  of  a  cover-glass,  is 
enclosed,  by  means  of  some  vaseline,  in  a  hollowed 
glass  slide  (vide  Technical  Appendix) .  Then  larger 
and  larger  quantities  of  the  disinfectant  are  added 
gradually  to  the  serum  tube,  and  after  each  addition 
a  drop  culture  is  again  made.  After  remaining  from 
twenty-four  to  forty-eight  hours  in  the  incubator, 
we  can  convince  ourselves  with  the  microscope  of  the 
growth  in  the  different  drops. 

In  case  of  a  degree  of  concentration  necessary  to 
antisepsis,  the  fungus  is  cultivated  in  bouillon  and 
10  c.c.  of  the  bouillon  (which  is  still  free  from  spores 
and  which  has  been  filtered  through  asbestos  in  order 
to  get  rid  of  any  clumps  of  bacilli  which  may  be 
present)  are  replaced  with  a  corresponding  amount 
of  the  disinfectant  solution.     From  these  tubes  we 


92  ATLAS   OF   BACTERIOLOGY. 

take,  at  the  end  of  one  minute,  five  minutes,  ten 
minutes,  fifteen  minutes,  thirty  minutes,  one  hour, 
etc.,  a  small  platinum  loop  full  of  material,  place  the 
latter  in  10  c.c.  of  lukewarm  liquefied  gelatin,  and  form 
plates.  We  then  obtain  statements  like  the  following : 
X  per  cent  of  the  disinfectant  proves  fatal  in  twenty 
minutes,  y  per  cent  in  one  minute,  etc.  If  we  sus- 
pect that  the  trace  of  disinfectant,  which  is  conveyed 
by  the  loop,  may  have  made  the  gelatin  aseptic  and 
may  thus  have  simulated  destruction  of  the  bacteria, 
we  should  make  a  control  inoculation  of  fresh  material 
in  gelatin  to  which  a  similar  trace  of  the  disinfecting 
fluid  has  been  added. 

The  disinfectant  to  be  tested  should  always  be  dis- 
solved in  water.  If,  on  account  of  the  slight  solubil- 
ity in  water,  the  use  of  alcohol  in  the  production  of 
the  original  solution  is  indispensable,  special  control 
experiments  are  necessary  in  order  to  show  that  the 
action  of  the  alcohol  was  not  injurious. 

When  using  nutrient  media  which  are  rich  in 
albumin,  we  require  much  larger  amounts  of  the 
disinfectant,  both  for  the  production  of  asepsis  as 
well  as  for  that  of  antisepsis,  than  when  using  media 
which  are  poor  in  albumin.*  Thus  in  bouillon 
creolin  produces  asepsis  when  present  in  the  propor- 
tion of  1 :  15000-1 :  5000 ;  in  beef  serum  only  in  the 
proportion  of  1 :  150.  In  bouillon  free  from  peptone 
or  containing  one  per  cent  peptone,  cholera  vibriones 
are  killed  in  one-half  hour  on  the  addition  of  0.01  per 
cent  HCl ;  on  the  addition  of  two  per  cent  peptone, 
only  when  0.04  per  cent  HCl  is  added.  For  diag- 
nostic purposes  we  will  usually  make  the  tests  in  one 
*  Phenol  is  said  to  be  an  exception. 


THE   VITAL   CONDITIONS   OF   BACTERIA.  93 

per  cent  peptone  solutions  if  we  do  not  wish  to  use 
one  of  the  non-albuminous  media  described  on  page 
86.  At  all  events,  the  bacteria  which  are  to  be  com- 
pared must  be  treated  in  exactly  the  same  way,  and, 
if  the  results  are  to  be  published,  the  various  condi- 
tions of  the  experiment  must  be  described  in  detail. 
Of  bacteria  which  are  free  from  spores  not  much  is 
known  concerning  their  varying  resistance  according 
to  variety  and  nutrient  medium,  but  a  few  statements 
in  this  regard  have  been  made  concerning  staphylo- 
cocci (Esmarch:  Z.  H.,  V.,  1889,  p.  72). 

A  combination  of  disinfectants  increases  their 
action.  In  particular,  the  addition  of  acid  (hydro- 
chloric or  tartaric  acid)  intensifies  the  effect  of  subli- 
mate, and  also  of  solutions  of  phenol  and  cresol. 
Moreover,  the  effect  is  more  certain  upon  a  few  than 
upon  many  germs,  and  greater  at  a  higher  than  at  a 
lower  temperature. 

4    DEFICIENCY  OF  FOOD  AND  WATER. 

If  bacteria  which  require  nutritious  substances  in 
order  to  thrive  are  placed  in  distilled  water,  they 
usually  die  rapidly  (within  a  few  days) .  In  spring 
water  (even  when  sterilized),  the  duration  of  life  is 
usually  not  more  than  eight  to  fourteen  days,  and 
proliferation  is  rare.  In  a  series  of  cases,  however, 
a  much  longer  duration  of  life  had  been  observed 
{vide  Loffler :  " Das  Wasser  u.  d.  Mikroorg.,"  Fischer, 
1896).  The  sensitiveness  of  bacteria  to  a  deficiency 
of  water  varies  greatly.  Upon  drying  nutrient  media 
growth  soon  ceases.  On  the  other  hand,  the  dura- 
tion of    life    upon    nutrient   media    (agar,   gelatin, 


94 


ATLAS   OF   BACTERIOLOGY. 


potato)  which  are  drying  slowly  at  the  temperature 
of  the  room,  is  often  astonishingly  long,  even  when 
this  cannot  be  attributed  to  the  development  of  endo- 
spores.  Even  after  the  lapse  of  a  year  it  is  found 
occasionally  that  such  a  shrivelled  remnant  of  a  cul- 
ture furnishes  the  most  beautiful  cultures  in  bouillon. 

The  question  has  often  been  investigated — and  with 
very  contradictory  results — as  to  the  length  of  the  time 
during  which  bacteria  free  from  spores,  which  have 
dried  upon  pieces  of  glass  or  threads  of  silk,  may 
remain  alive.  We  know  now  that  this  is  influenced 
by  numerous  factors.  An  idea  of  their  viability  is 
furnished  by  the  following  table  of  Sirena  and  Alessi 
(C.  B.,  XL,  484). 

In  bouillon  cultures  free  from  spores  or  in  watery 
deposits  of  bacteria  silk  threads  were  dipped,  and 
part  of  them  were  placed  in  test  tubes  one-third  full 
of  sulphuric  acid  or  calcium  chloride,  part  were  al- 
lowed to  dry  in  the  open  air  under  various  conditions. 


Period  at  which  Death  Occurred 
(in  Days). 

On  drying. 

3  03 

^1 

a  . 
II 

5*   03   CO 

S3  O  0) 

5  o  * 

0. 

03  O 
O 

t-H 

Vibrio  cholerce  asiaticae 

Bacterium  cholerae  gallinarum 
Bacterium  typhi 

1 

2 

41 

35 

63 

114 

1 
1 
1 

44 
53 
31 

1 

1 

18 

31 

31 

131 

1 

5 

64 

5 

164 

12 
59 

68 

Bacterium  mallei 

Bacterium  erysipelatos  suum 
Streptococcus  lanceolatus 

59 

192 

Cholera  vibriones   are  especially  well   known  for 
their  slight  power  of  resistance  to  desiccation.     Ex- 


THE   VITAL   CONDITIONS   OF   BACTERIA.  95 

tended  experiments  of  tlie  authors  just  mentioned 
put  their  duration  of  life  at  from  one  to  five  hours 
according  to  the  mode  of  desiccation  (the  results  are 
similar  to  those  of  E.  Koch  in  his  first  experiments). 
But  it  is  evident  from  the  results  obtained  by  all 
writers  that  desiccation  experiments  must  be  espe- 
cially varied  and  many-sided,  if  they  are  to  be  con- 
vincing. The  surprising  result  has  recently  been 
obtained  in  regard  to  very  many  varieties  which  are 
sensitive  to  desiccation  (this  is  true  particularly  of 
the  cholera  vibriones)  that,  under  certain  circum- 
stances, they  may  remain  alive,  when  dried,  for  a 
much  longer  time.  Thus  Koch  found  the  duration 
of  life  to  be  a  few  hours;  Kitasato  (Z.  H.,  Y.,  135) 
fourteen  days;  French  writers  and  Berckholz  (A.  G. 
A.,  Y.,  1)  found  it  one  hundred  and  fifty  to  two  hun- 
dred days  under  specially  favorable  conditions.  Ac- 
cording to  most  writers  these  favorable  conditions  in- 
clude: stay  in  the  desiccator,  removal  from  agar  or 
potato  cultures  instead  of  bouillon  cultures,  the  use 
of  silk  threads  instead  of  pieces  of  glass.  Special 
mention  must  also  be  made  of  the  fact  that  in  none 
of  these  experiments  could  anything  of  the  nature  of 
spores  (arthrospores)  be  positively  recognized. 

5.  RELATIONS  TO  OXYGEN  AND  SOME  OTEHR  GASES. 

In  their  relations  to  oxygen  bacteria  are  divided 
usually  into  three  classes  (Fliigge  and  Liborius) : 

/.  Strict  Aerobics. — Growth  occurs  only  when  the 
air  finds  access;  any  obstruction  to  the  latter  inter- 
feres with  the  growth.  Free  oxygen  is  particularly 
necessary  to  the  development  of  spores. 


96  ATLAS   OF   BACTERIOLOGY. 

//.  Strict  Anaerobics.  —  Growth  and  sporulation 
take  place  onl^^  during  complete  exclusion  of  oxygen. 
This  class  includes  bacillus  oedematis  maligni,  bacil- 
lus tetani,  bacillus  Chauvoei,  and  a  large  number  of 
inhabitants  of  slime  and  earth.  When  exposed  to  the 
free  oxygen  of  the  atmosi)here  the  vegetative  forms  of 
these  bacteria  perish  very  readily,  but  the  spores  ex- 
hibit great  resistance  to  oxygen.  As  the  anaerobics 
are  excluded  from  the  main  supply  of  energy  which 
is  at  the  command  of  aerobic  bacteria  (oxidation  of 
the  absorbed  nutritive  material  by  means  of  free  oxy- 
gen) ,  they  must  rely  upon  nutritive  substances  which 
possess  great  potential  energy  and  which,  like  grape 
sugar,  for  example,  set  free  energy  (heat)  by  sei)ara- 
tion  into  two  smaller  molecules  (for  exami:>le,  alcohol 
and  carbonic  acid ;  or  acetic  and  lactic  acids) .  Hence 
anaerobics  are  almost  always  cultivated  upon  gelatin 
or  agar  which  contains  one  to  two  per  cent  of  grape 
sugar. 

III.  Facultative  Aerobics  and  Facultative  Anae- 
robics.— The  large  majority  of  the  bacteria  which,  as 
a  rule,  are  cultivated  aerobic  (including  almost  all 
the  pathogenic  forms)  tolerate  a  restriction  in  the 
supply  of  oxygen  without  suffering  injury  or  ex- 
hibiting diminished  growth.  In  many  cases  life 
in  the  animal  body,  for  example  in  the  intestinal 
canal,  decidedly  involves  a  diminution  or  aboli- 
tion of  the  supply  of  oxygen.  When  oxygen  is 
excluded  the  formation  of  pigment  is  almost  al- 
ways abolished,  while  virulent  products  of  dis- 
assimilation  are  produced  in  greater  abundance 
(Hiippe). 

It  is  a  very  important  fact  that  recent  investigations 


THE  VITAL  CONDITIONS   OF   BACTEBIA.  97 

have  shown  that  aerobic  races  exist  among  the  anae- 
robic varieties. 

It  is  observed  not  very  rarely  that  varieties  which 
on  isolation  exhibited  more  or  less  anaerobic  growth 
(for  example,  grew  chiefly  into  the  depth  of  the  agar 
stick  canal),  in  time  manifest  a  purely  aerobic  con- 
dition, i.e.,  distinct  growth  upon  the  surface  and 
dwarfed  growth  in  the  canal. 

These  observations  show  that  two  varieties  cannot 
be  distinguished  from  one  another  by  simply  calling 
one  aerobic,  the  other  anaerobic. 

In  addition  to  the  strict  anaerobics  all  the  faculta- 
tive anaerobic  varieties  thrive  well  in  nitrogen  and 
hydrogen,  but  they  tolerate  carbonic  acid  in  various 
ways. 

A  large  number  do  not  flourish  at  all,  but  their 
development  is  entirely  checked  until  oxygen  is  again 
supplied— for  example,  bacillus  anthracis,  bacillus 
subtilis,  and  allied  forms.  Of  several  varieties  (an- 
thrax, cholera)  it  has  been  ascertained  that  the  major- 
ity of  individuals  are  killed  very  quickly  by  carbonic 
acid,  while  certain  ones  oif  er  a  very  vigorous  resistance 
and  render  complete  sterilization  by  CO^  impossible. 
A  second  group  exhibits — especially  when  the  ex- 
periment is  made  at  incubating  temperature — feeble 
growth  (staphylococci,  streptococci),  while  a  third 
group  is  not  at  all  injured  (bacterium  prodigiosum, 
bacterium  acidi  lactici,  bacterium  typhi).  These 
grow  as  well  as  they  do  in  the  air,  and  the  liquefac- 
tion of  the  gelatin  is  not  interfered  with.  As  a  matter 
of  course,  pigment  is  not  formed  on  account  of  the 
absence  of  oxygen.  A  mixture  of  twenty -five  per  cent 
air  with  seventy-five  per  cent  carbonic  acid  exerts  no 
7 


98  ATLAS   OF   BACTERIOLOGY. 

demonstrable  injurious  influence  upon  bacteria  which 
remain  absolutely  undeveloped  in  pure  carbonic  acid 
(C.  Fraenkel:  Z.  H.,  Y.) 

Sulphuretted  hydrogen  in  large  amounts  is  al- 
ways an  active  bacterial  poison;  small  amounts  kill 
the  bacterium  Pfliigeri  very  rapidly  (Lehmann  and 
Tollhausen:  C.  B.,  V.,  785). 

6.    INFLUENCE  OF  TEMPERATURE  ON   THE   LIFE  OF 
BACTERIA. 

Each  variety  of  bacteria  makes  certain  demands 
upon  the  temperature  of  its  nutritive  medium.  Vege- 
tative bacterial  life  is  possible  from  0°  to  about  70°, 
but  there  are  some  varieties  which  flourish  at  the 
lower  range,  others  at  the  upper  range.  In  each 
variety  the  minimum  and  maximum  of  temperature 
are  separated  by  about  30°,  and  the  following  com- 
prehensive classification  may  be  made  according  to 
the  temperature  requirements : 

PsychropMlic  bacteria :  minimum  at  0°,  best  at  15°- 
20°,  maximum  at  about  30°.  These  varieties  usually 
live  in  the  water.  They  include,  for  example,  many 
phosphorescent  bacteria  of  the  ocean  (vide  Forster : 
C.  B.,  XII.,  431). 

Mesophilic  bacteria:  minimum  at  10°-15°,  best  at 
37°,  maximum  at  about  45°.  These  include  all  the 
pathogenic  varieties,  because  acclimatization  to  the 
bodily  temperature  is  a  necessary  condition  of  their 
pathogenic  action. 

Bacillus  vulgatus,  *  which  still  thrives  at  50°,  fur- 
nishes a  transition  to  the  following  group. 

*  Bacillus  vulgatus  thrives  at  from  15°-50°,  and  odg  variety  of 
Globig's  ranges  from  5r-68°,  but  such  cases  are  very  rare.  Glo- 


THE    VITAL   CONDITIONS   OF   BACTERIA.  99 

Thermophilic  bacteria:  minimum  at  40°-49°,  best 
at  50°-55°,  maximum  at  GO'^-TO".  These  include  many 
bacteria  of  the  soil,  and  almost  all  the  sporulating 
bacilli  related  to  bacillus  mesentericus.  According  to 
Globig  about  thirty  varieties  are  still  capable  of  de- 
velopment at  60°,  and  a  few  at  70°  (Z.  H.,  III.,  294). 
Miquel  {Ann.  de  Micrograph.,  I.,  4;  C.  B.,  V.,  281) 
has  described  a  bacillus  thermophilus  Miqu. ,  which 
thrives  at  from  42°-72°,  best  at  65°-70°,  and  has  its 
habitat  in  i)rivies,  the  intestinal  contents,  and  dirty 
water.  The  description  is  insufficient  to  distinguish 
the  bacillus. 

Kecently  Lydia  Rabinowitsch  has  described  eight 
thermophilic  facultative  anaerobic  varieties;  they 
were  all  non-motile  sporulating  rods*,  which  throve 
best  at  60°-70°,  but  proliferated  slowly  even  at  34°-44°, 
best  in  an  anaerobic  agar  culture  (Z.  H.,  XX.,  163). 
These  varieties  are  widely  diffused,  particularly  in 
the  faeces,  but  Rabinowitsch  did  not  make  any  com- 
parison with  the  forms  described  by  previous  writers. 

By  gradually  increasing  and  lowering  the  temper- 
ature Dieudonne  (C.  B.,  XVI.,  965)  succeeded  in  in- 
creasing the  temperature  interval  within  which  the 
bacillus  anthracis  is  capable  of  proliferating.  The 
bacillus  could  be  adapted  gradually  to  a  temperature 
of  42°.  According  to  the  assumption  of  some  writers 
pigeons  are  tolerably  immune  to  ordinary  anthrax  on 
account  of  their  high  temperature  (42°),  but  when 
the  bacilli  had  been  adapted  to  high  temperatures 
the  pigeons  died  more  frequently  after  inoculation. 

Still  more  striking;; Tfcre , the  results  Vh^n  DiQudonne 

big  found  unusuaDy  narrow  ranges  for  many  thermopuile  varie- 
ties; for  example,  oueiorm  §rew  pirly  at;'bet\^?('ji^r'\65*.\  ' 


100  ATLAS   OF   BACTERIOLOGY. 

gradually  acclimatized  the  bacilli  to  a  temperature  of 
12°  and  showed  that  they  could  then  kill  frogs  which 
are  kept  at  12°. 

Temperatures  somewhat  below  the  minimum  for 
the  variety  in  question  inhibit  the  development  but 
are  not  otherwise  injurious.  Petruschky  has  recently 
recommended  keeping  them  in  an  ice-box  (about 
4°-6°).  He  claims  that  in  this  way  varieties  which 
perish  easily  can  be  kept  not  only  alive  and  capable 
of  proliferation  but  also  virulent,  after  they  have 
been  allowed  to  grow  for  two  days  at  a  temperature 
of  20°  (streptococci,  etc.). 

Temperatures  below  0°  also  act  very  slowly  and 
injure  the  different  varieties  with  varying  rapidity. 

If  temperatures  5° -10°  above  the  best  act  upon  the 
culture,  the  latter  is  injured  in  various  ways.  Eaces 
of  diminished  intensity  of  growth  develop,  the  viru- 
lence and  fermentative  power  diminish,  and  the  capa- 
bility of  sporulation  is  gradually  lost.  The  injurious 
influence  sometimes  predominates  in  one  direction, 
sometimes  in  another.  If  the  maximum  temperature 
is  exceeded,  the  culture  dies.  For  the  psychrophilic 
forms  about  37°,  for  the  mesophilic  forms  about  60°, 
for  the  thermophilic  forms  75°,  are  quite  rapidly 
fatal  temperatures.  No  bacterium  free  from  spores 
can  tolerate  a  temperature  of  100°  even  for  a  few 
minutes. 

7.  MECHANICAL  AND  ELECTRICAL    EFFECTS. 

OiIj:  xuttur^ea-  are.  jr^a^e,  almgst  exiclusively  upon 
nutri^nffc. j^iedi^.^hicJi.aiTe  k^eptrquje;;  (it  is  only  to 
secjaEOr  abundant  sporulation  in  ^uid  media,  in  the 


THE  VITAL  CONDITION'S  OF   BACTERIA.  101 

case  of  aerobic  varieties,  that  a  slight  movement  of 
the  fluid  is  usually  secured).  Hence  a  theoretical  in- 
terest alone  attaches  to  the  fact  that,  according  to 
Meltzer's  recent  investigations,  brief  or  feeble  shak- 
ing of  bacteria  cultures  in  vessels  one-third  full  acts 
favorably  on  the  development  of  the  bacteria,  while 
constant  and  vigorous  shaking  for  a  number  of  hours, 
especially  when  balls,  of  glass  are  placed  in  the  fluid, 
scatters  the  bacteria  into  a  fine  dust  and  kills  them. 
The  various  bacteria  act  in  different  ways  (Ztschr.  f. 
Biolog.,  XXX.,  p.  454). 

Meltzer  makes  the  very  remarkable  statement  that 
the  feeble  tremor  which  a  steam  engine  running  day 
and  night  communicated  to  the  floor  of  a  brewery 
was  sufficient  to  kill,  in  four  days,  all  the  germs  of 
bacillus  mycoides  and  subtilis  kept  in  a  bottle  of 
nutrient  fluid. 

Concerning  our  scanty  knowledge  of  the  influence 
of  the  electrical  current  upon  bacteria,  vide  Frieden- 
thal:  C.  B.,  Part  L,  XIX.,  319. 

The  majority  of  the  effects  of  the  electrical  currents 
hitherto  observed  are  readily  explained  by  the  action 
of  heat  and  electrolysis. 

8.   EFFECT  OF  LIGHT. 

The  development  of  many  bacteria,  perhaps  of  the 
majority,  is  impeded  by  the  action  of  diffuse  daylight 
upon  the  cultures,  and  still  more  by  the  action  of 
direct  sunlight.  After  a  time  the  bacteria  lose  the 
power  of  proliferating  freely  in  the  dark  and  we  ob- 
tain a  generation  of  feeble  organisms ;  for  example, 
they  liquefy  imperfectly,  form  pigment  imperfectly, 


102  ATLAS  OF  BACTERIOLOGY. 

are  less  pathogenic,  etc.  It  is  only  after  repeated 
transference  to  fresh  nutrient  media  in  the  dark  that 
they  regain  their  old  power.  When  the  action  of 
light  is  still  more  prolonged  the  micro-organisms  die. 
In  order  to  test  the  sensitiveness  to  light  it  is  best, 
according  to  H.  Buchner,  to  expose  to  diffuse  light 
or  to  sunlight  densely  crowded  plates  of  gelatin  or 
agar,  a  black  paper  cross  being  pasted  on  the  light 
side.  In  order  to  exclude  the  action  of  heat  the  light 
may  first  be  passed  through  a  layer  of  water  or 
alum  a  few  centimeters  in  thickness.  After  exposure 
to  the  light  for  one-half,  one,  one  and  a  half,  two 
hours,  etc. ,  the  plates  are  placed  in  the  dark  and  it  is 
noted  whether  the  bacteria  develop  only  at  the  loca- 
tion of  the  cross.  When  all  the  colonies  which  were 
illuminated  have  perished,  we  find  a  sharply  defined 
cross,  formed  of  cultures  in  a  light  field. 

During  March,  July,  and  August  bacteria  putidum 
and  prodigiosum  are  killed  in  one-half  hour  by  direct 
sunlight.  In  November,  at  the  end  of  one  and  a  half 
hours,  their  power  of  producing  pigment  and  tri- 
methylamin  is  interfered  with  materially,  they  grow 
slowly,  and  bacterium  prodigiosum  liquefies  poorly. 
The  organisms  died  in  one  and  a  half  and  two  and  a 
half  hours. 

In  diffuse  daylight,  inhibition  of  development  oc- 
curs in  the  spring  and  summer  in  three  and  a  half 
hours,  in  winter  in  four  and  a  half  hours;  death 
occurs  in  from  five  to  six  hours.  The  electric  arc 
light,  of  900  candle  power,  inhibited  development  in 
^ye  hours,  and  killed  the  germs  in  eight  hours. 
Bacterium  coli,  bacterium  typhi,  and  bacillus  anthra- 
cis  reacted  in  a  similar  manner. 


THE   VITAL   CONDITIONS   OF   BACTERIA.  103 

The  ultra  violet,  violet  and  blue  light  have  a  power- 
ful injurious  effect,  green  light  has  a  feeble  effect, 
and  red  and  yellow  have  none  at  all. 

The  action  of  light  seems  to  be  dependent  in  part 
on  the  oxygen  of  the  air.  Strict  anaerobic  (tetanus) 
and  facultative  anaerobic  varieties  (bacterium  coli) 
tolerate  sunlight  very  well  if  there  is  complete  exclu- 
sion of  oxygen. 

Richardson  and  recently  Dieudonne  have  discov- 
ered a  fact  which  possesses  great  importance  in  re- 
gai-d  to  the  mechanism  of  the  action  of  light,  al- 
though it  does  not  explain  everything.  They  found 
that  hydrogen  hyperoxide  (H^OJ  develops  in  a  short 
time  (in  ten  minutes  in  direct  sunlight)  upon  illumi- 
nated agar  plates,  but  only  in  blue  to  ultra  violet 
light.  *  An  agar  plate,  half  covered  with  black  paper, 
is  exposed  to  the  light,  then  a  paste  containing  a 
small  amount  of  potassium  iodide  is  poured  over  it 
and  this  followed  by  a  weak  solution  of  sulphate  of 
ferric  oxide,  the  illuminated  side  turns  a  bluish-black. 
In  gases  which  contain  no  oxygen  H.,02  does  not  form 
and  light  does  not  give  rise  to  any  injury.  This 
also  explains  the  fact  that  slight  "  attenuation"  of  the 
bacilli  is  also  observed  frequently  when  agar  plates 
which  have  been  standing  in  the  sun  f  are  inocu- 
lated. Bacteria  which  have  been  previously  ex- 
posed to  the  light  develop  with  special  difficulty  on 
an  illuminated  nutrient  medium. 

*  Hours  elapse  before  H2O2  can  be  demonstrated  upon  gelatin. 

f  Other  decompositions  of  the  nutrient  media  by  sunlight  may 
interfere  occasionally  with  the  subsequent  growth  of  bacteria, 
for  example,  the  development  of  formic  acid  from  tartaric  acid 
(Duclaux) . 


104  ATLAS   OF   BACTERIOLOGY. 


9.    EFFECT  OF  OTHER  BACTERIA  UPON  BACTERIAL 
GROWTH. 

Althougli  it  is  the  object  of  every  bacteriologist  to 
obtain  only  pure  cultures,  it  must  not  be  forgotten 
that  in  nature  bacteria  often  appear  in  mixed  cul- 
tures. When  water,  milk,  the  intestinal  contents  of 
sick  or  healthy  individuals,  etc.,  are  examined,  sev- 
eral varieties  will  always  be  found  at  the  same  time. 
Although  this  admixture  usually  appears  to  be 
purely  accidental,  it  is  found  on  closer  investigation 
that,  in  the  domain  of  bacteriology,  there  are  syn- 
ergetic  (favoring  the  growth  of  one  another)  and 
antagonistic  (injuring  one  another)  varieties.  Nencki 
speaks  of  symbiosis  and  enantobiosis. 

Garre  demonstrated  the  antagonism  experimentally 
by  making  streak  cultures  of  various  bacteria  upon 
gelatin  plates,  in  the  shape  of  parallel  or  intersect- 
ing lines.  It  was  then  found  that  certain  varieties 
thrive  very  poorly  or  not  at  all  when  another  variety 
is  growing  in  their  immediate  neighborhood.  In 
very  many  cases  the  antagonism  is  one-sided.  For 
example,  bacterium  putidum  grows  very  well  when 
inoculated  between  closely  approximated,  well-devel- 
oped streaks  of  staphylococci.  On  the  other  hand, 
micrococcus  pyogenes  does  not  grow  when  inoculated 
between  luxuriantly  developing  cultures  of  bacterium 
putidum,  and  the  former  remains  very  meagre  when 
both  varieties  are  applied  in  streak  cultures  at  the  same 
time  (Garre:  Corresp.  f.  Schweizer  Aerzte,  1887). 

Or  we  make  plates  of  gelatin  or  agar  (for  liquefy- 
ing varieties)  which  have  been  infected,  in  the  melted 


THE  VITAL  CONDITIOl^S  OF   BACTERIA.  105 

condition,  with  an  equal  number  of  individuals  of  two 
different  varieties  of  bacteria.  In  many  cases  only 
one  variety  will  undergo  development  (Lewek:  C.  B., 
VII.,  107). 

The  following  is  the  third  method  of  making  the 
experiment.  The  same  fluid  nutrient  medium  is  in- 
oculated with  two  varieties  and  later  we  ascertain  the 
victor  in  the  struggle,  either  with  the  microscope  or 
macroscopically  upon  thin  plates.  To  this  category 
belongs  the  frequent  experience  that  fermentation- 
producers,  when  present  in  large  numbers  in  a  suit- 
able medium,  prevail  over  contaminating  bacteria. 
The  latter  sometimes  disappear  entirely. 

The  following  practical  inference  may  be  drawn 
from  these  experiences.  In  counting  bacteria  very 
dense  plates  may  not  be  regarded  as  decisive,  and  in 
the  isolation  of  certain  varieties  thin  plates  may  also 
be  necessary.  For  example,  in  isolating  bacterium 
Pfliigeri  from  an  abundance  of  bacterium  putidum; 
no  bacteria  Pfliigeri  grow  within  a  circle  of  several 
millimetres  around  each  culture  of  bacterium  putidum 
(K.  B.  Lehmann). 

Finally,  bacteria  may  antagonize  one  another  with- 
in the  animal  body.  As  Emmerich  showed,  animals 
infected  with  anthrax  may  be  saved  by  subsequent 
inoculation  with  streptococcus  pyogenes.  It  is  im- 
possible to  enter  into  the  mechanism  of  this  process 
within  the  limits  of  this  work. 

Greater  practical  importance  attaches  to  the  sym- 
biosis of  bacteria,  as  is  shown  by  the  following 
examples. 

1.  A  series  ol  bacteria  thrive  better  in  company 
with   others   than  alone.      Certain  anaerobics  even 


106  ATLAS   OF   BACTEKIOLOGT. 

thrive  on  the  admission  of  air,  if  other  aerobic  varie- 
ties are  present  {vide  bacillus  tetani). 

2.  Certain  chemical  actions,  for  example,  the  de- 
composition of  nitrate  into  gaseous  nitrogen  cannot 
be  effected  by  some  bacteria  alone,  while  it  can  be 
done  by  two  forms  in  combination.  This  experience 
is  to  be  remembered  in  looking  for  the  xjrodncers  of 
certain  decompositions.  When  the  isolated  varieties 
do  not  act  singly  or  act  incompletely,  combinations 
must  be  examined. 

3.  In  a  similar  way  it  has  been  observed,  for  ex- 
ample, that  among  a  series  of  soil  bacteria  each 
single  variety  is  not  pathogenic,  while  certain  com- 
binations, when  introduced  into  the  animal,  make 
the  latter  sick.  This  experience  also  merits  special 
attention  in  the  search  for  the  producers  of  a  new  or 
obscure  disease. 

Some  writers  also  assume  the  production  of  cholera 
by  two  germs  (diblastic  theory). 

4.  Feeble  pathogenic  varieties  (for  example,  atten- 
uated tetanus  bacilli)  are  said  to  gain  in  virulence 
when  cultivated  with  bacterium  vulgare. 


D.  The  Conditions  of  Formation  and  Germi- 
nation of  Spores. 

Biological  Characters  of  Spores. 

The  extent  of  the  formation  of  endogenous  spores 
appears  to  be  imperfectly  known  at  the  present  time. 
Apart  from  a  large  group  of  bacilli  which  are  re- 
lated to  bacillus  anthracis  and  bacillus  tetani,  un- 
doubted endogenous  spores  are  known  only  in  sarcina 


FORMATION   AND   GERMINATION   OF   SPORES.       107 

pulmonum  and  the  peculiar  spirillum  endoparagoci- 
cum. 

As  H.  Buchner  (C.  B.,  YIII.,  1)  showed,  the  for- 
mation of  spores  takes  i^lace  in  suitable  varieties 
when  the  nutrient  medium  is  beginning  to  be  ex- 
hausted, i.e.,  it  is  most  rapid  in  very  poor  media. 

On  the  other  hand,  a  good  nutrient  medium  not 
alone  facilitates  the  development  of  the  bacilli  but 
also  that  of  the  spores,  in  so  far  as  the  vigorously 
growing  bacilli  also  sporulate  luxuriantly  and  con- 
stantly. The  crop  of  spores  is  disproportionately 
large.  Whether  the  quality  (power  of  resistance)  of 
the  spores,  which  grow  upon  different  nutrient  media, 
also  differs,  does  not  seem  to  have  been  investigated 
methodically. 

The  temperature  must  sometimes  (always  ?)  be 
higher  for  sporulation  than  for  vegetative  growth. 
For  example,  the  bacillus  anthracis  flourishes  at 
13°-14°,  but  does  not  form  spores  under  18°. 

All  aerobic  bacteria  require  the  entrance  of  oxygen 
particularly  for  sporulation.  The  mode  in  which 
facultative  anaerobic  varieties  act  has  not  been  as- 
certained. 

Strict  anaerobics  produce  spores  only  on  the  exclu- 
sion of  oxygen  or  on  the  admission  of  oxygen  in 
mixed  cultures  or  when  synergetic  bacteria  have 
perished. 

Spores  never  germinate  in  the  exhausted  nutrient 
medium  in  which  they  have  been  formed,  or  which 
has  been  affected  injuriously  by  the  products  of  dis- 
assimilation.  It  is  only  after  removal  to  a  new  nu- 
trient medium  that  germination  takes  place  (the  mor- 
phological details  have  been  described  on  page  79) . 


108  ATLAS   OF   BACTERIOLOGY. 

Spores  are  much  more  resistant  than  vegetative 
forms  to  all  injurious  influences.  They  require  no 
nourishment  or  water  in  order  to  remain  capable  of 
germination  for  years  and  decades,*  they  are  much 
more  indifferent  to  gases  than  bacilli,  and  the  spores 
of  anaerobic  varieties  usually  tolerate  free  oxygen 
welLt 

The  power  of  resistance  of  the  spores  to  dry  and 
moist  heat  is  very  considerable.  Dr^^  heat  is  toler- 
ated relatively  very  well,  and  many  spores  resist  a 
temperature  of  100°.  In  the  moist  condition  a  tem- 
perature of  70°  kills  the  anthrax  bacillus  in  one 
minute,  while  the  spores  resist  this  temperature  for 
hours,  and  in  water  or  steam  at  100°  they  live  from  two 
to  ^Ye  minutes,  occasionally  even  from  seven  to  twelve 
minutes.  The  varying  resistance  of  different  anthrax 
spores  (v.  Esmarch:  Z.  H.,  Y.,  p.  67)  seems  to  be 
partly  a  race  peculiarity.  It  is  very  probable,  more- 
over, that  the  nutrient  medium,  the  temperature  at 
the  formation  of  the  spores,  the  degree  of  maturity, 
etc.,  also  exert  an  influence  upon  the  resistance. 
Careful  investigations  on  this  subject  are  almost  en- 
tirely lacking,  but  Percy  Frankland  has  shown  that 
spores  formed  at  20°  are  more  resistant  to  light  than 
those  formed  at  incubation  temperature  (C.  B.,  XV., 
p.  110). 

*  According  to  an  observation  of  v.  Esmarch  the  virulence  of 
anthrax  spores  seems  to  be  lost,  in  the  course  of  time,  before 
their  power  of  germination. 

f  Dry  garden  earth  containing  the  spores  of  malignant  oedema 
preserved  the  latter  excellently  in  my  laboratory  for  four  y^ars. 
On  the  other  hand,  tetanus  spores  which  were  dried  on  threads 
and  kept  in  the  room  had  perished  at  the  end  of  three  days  ; 
they  were  still  alive  on  the  second  day. 


FORMATION-   AND   GERMINATION"   OF   SPORES.       109 

The  resistance  is  tested  by  simply  hanging  in  the 
steam  chamber  little  tulle  bags  containing  fragments 
or  bits  of  glass  upon  which  anthrax  spores  have  been 
dried.  From  minute  to  minute  a  bag  is  removed  and 
the  bits  of  glass  placed  upon  an  agar  plate  which  is 
kept  at  incubating  temperature.  Anthrax  spores  are 
obtained  by  careful  removal  of  sporulating  agar  streak 
cultures,  and  warming  the  emulsion,  prepared  with 
little  water,  to  70°  for  five  minutes. 

The  varying  resistance  of  apparently  identical  an- 
thrax spores  possesses  great  practical  importance: 
(1)  in  disinfection  tests  which  may  be  made  only 
with  spores  of  known  resistance ;  (2)  in  differential 
diagnosis,  because  it  shows  that  we  must  be  on  our 
guard  against  creating  two  species  based  on  a  differ- 
ence in  resistance. 

Various  forms  which  occur  in  hay  and  soil  possess 
remarkable  resistance. 

Christen  found  (C.  B.,  XVII.,  p.  498),  for  example, 
that  in  steam  under  pressure  the  resisting  spores  of 
the  soil  required  for  their  destruction :  At  100°,  more 
than  sixteen  hours;  105°-110°,  two  to  four  hours; 
115°,  thirty  to  sixty  minutes;  125°-130°,  five  minutes 
or  more;  135°,  one  to  five  minutes ;  140°,  one  minute. 
The  apparatus  raised  objects  very  rapidly  to  the  de- 
sired temperature. 

Spores  are  also  very  resistant  to  chemical  agents. 
Thus,  anthrax  spores  require,  according  to  their  origin 
(v.  Esmarch :  I.  c.)  a  five-per-cent  solution  of  carbolic 
acid  at  least  two  days,  in  some  cases  even  forty  days. 
A  one-per-cent  aqueous  solution  of  corrosive  subli- 
mate is  withstood  by  very  resistant  anthrax  spores  as 
much  as  three  days,  although  their  virulence  was  lost 


110  ATLAS   OF   BACTERIOLOGY. 

in  twenty  hours.  These  tests  are  made  best  with  thin 
deposits  of  the  spores  in  water,  to  which  the  disin- 
fectant is  added,  as  we  have  indicated  above  in  regard 
to  the  tests  of  antiseptic  action  against  bacilli. 

In  order  to  test  the  resistance  of  spores  to  gases  it 
is  best  to  dry  them  upon  pieces  of  glass ;  the  gases 
are  allowed  to  act  first  in  a  dry  chamber,  then  in  one 
saturated  with  water. 

Spores  are  also  less  damaged  by  light  than  bacilli 
are;  as  in  the  case  of  bacilli  an  oxygenated  atmos- 
phere is  necessary  in  order  to  produce  injury  by 
light.  Anthrax  spores  on  agar  plates  were  found  by 
Dieudonne  to  be  killed  by  direct  sunlight  in  three 
and  a  half  hours  (bacilli  in  one  and  a  half  hours) ; 
when  oxygen  was  excluded  they  were  not  injured  by 
exposure  for  nine  hours. 

E.  The  Effects  of  Bacteria,  Especially  in  Re- 
gard to  Their  Employment  for  Diagnostic 
Purposes. 

The  effects  of  bacteria*  in  vitro  may  be  classified 
as  (1)  mechanical;  (2)  thermal;  (3)  optic;  and  (4) 
chemical.  They  will  be  discussed  in  this  order  and 
a  fifth  section  will  deal  with  the  effects  of  bacteria 
upon  the  living  animal  body  and  will  explain  the 
guiding  principles  necessary  to  the  comprehension  of 

*  It  goes  without  saying  that  a  classification  of  bacteria  into 
zymogenous,  saprogenous,  chromogenous,  and  pathogenic,  is  no 
longer  admissible.  For  example,  bacterium  coli  produces  fer- 
mentation in  solutions  of  sugar,  indol  and  sulphuretted  hydrogen 
in  albuminous  media,  brownish -yellow  foci  upon  potatoes,  and  is 
pathogenic  to  guinea-pigs,  i.e.,  it  combines  the  characteristics 
of  all  four  groups. 


THE  EFFECTS  OF  BACTERIA.  Ill 

their  pathogenic  iDfluence,  the  struggle  between  the 
bacteria  and  the  tissue  cells. 

All  the  effects  of  bacteria  depend:  (1)  upon  the 
present  condition  of  the  bacteria;  (2)  upon  the  nu- 
trient medium ;  (3)  upon  the  entrance  of  air ;  (4)  upon 
the  temperature;  and  (5)  upon  the  illumination.  A 
large  number  of  other  circumstances — in  part  less  im- 
portant, in  part  imperfectly  known — also  appear  to 
play  a  part. 

As  the  most  important  points  in  reference  to  tem- 
perature and  illumination  have  already  been  given, 
I  will  discuss  chiefly  the  influence  of  the  nutrient 
medium  and  the  entrance  of  air  on  the  one  hand, 
and  the  composition  of  the  terminal  culture  on  the 
other  hand.  Emphasis  must  be  constantly  laid  upon 
the  latter  point  in  order  to  show  as  clearly  as  pos- 
sible how  much  the  effects  of  bacteria  vary  according 
as  they  are  examined  in  a  fully  virulent  zymogenic, 
chromogenic,  or  pathogenic  condition,  or  in  an  attenu- 
ated condition. 

1.    MECHANICAL  EFFECTS. 

Under  the  microscope  it  is  readily  seen  that  many 
bacteria  exhibit  a  pronounced  active  movement,  and 
the  study  of  flagella  proves  that  almost  all  the  mo- 
tile varieties*  present  flagella  and  move  by  means  of 
these  appendages.  The  movement  varies  greatly  in 
character;  for  example,  creeping  (bacillus  megathe- 
rium), waddling   (bacillus  subtilis),  sinuous  (vibri- 

*  In  the  actively  motile  spirochsBte  Obermeieri  and  the  slowly 
creeping  beggiatoa  flagella  have  not  been  demonstrated,  so  that 
the  motion  is  supposed  to  be  due  to  an  undulating  narrow  mem- 
brane which  encloses  the  organism. 


112  ATLAS   OF   BACTERIOLOGY. 

ones).  It  is  sometimes  very  slow,  sometimes  so 
rapid  that  observations  in  detail  are  hardly  possible 
(bacterium  typhi) . 

In  some  cases  it  is  difficult  to  decide  whether  there 
is  a  real  active  movement  or  whether  the  micro-organ- 
isms do  not  exhibit  an  unusual  degree  of  the  so- 
called  Brownian  molecular  movement — i.e.,  the  danc- 
ing and  trembling  which  are  also  found  in  finely 
divided,  non-organized  particles.  In  such  cases, 
apart  from  the  attempt  to  render  the  flagella  visible, 
it  is  well  to  examine  the  organism  in  a  drop  of  five- 
per-cent  carbolic  acid  or  one-per-cent  corrosive  subli- 
mate. If  the  movements  then  continue,  we  have  had 
to  deal  only  with  molecular  movements.  Some 
varieties  do  not  always  exhibit  movements  of  their 
own,  but  they  may  be  absent  in  certain  nutrient 
media.  According  to  A.  Fischer  the  vital  movements 
may  be  lacking,  although  the  flagella  are  perfectly 
developed — for  example,  in  bacillus  subtilis  upon  a 
nutrient  medium  containing  two  to  four  per  cent 
ammonium  chloride.  In  two  different  cultures  of 
micrococcus  agilis  Ali-Cohen,  drawn  from  a  good 
source,  we  saw  neither  vital  movements  nor  flagella, 
and  reached  the  conclusion  that  the  same  variety 
may  occur  with  or  without  flagella. 

Certain  chemical  substances  attract  bacteria  (posi- 
tive chemotaxis),  others  repel  them  (negative  chemo- 
taxis).  Oxygen  in  particular  attracts  aerobic,  and 
repels  anaerobic  bacteria.  As  Beyerinck  showed, 
very  beautiful  chemotaxic  or  aerotaxic  figures  can  be 
obtained  in  the  following  way  :  In  a  test  tube  filled 
three-quarters  full  with  sterilized  water  is  placed  an 
unsterilized  bean,  pea,  or  the  like.     By  diffusion  the 


THE   EFFECTS   OF   BACTERIA.  113 

bean  gives  off  nutritive  substances,  which  slowly  ex- 
tend ui^ward.  In  this  feeble  nutrient  solution  cer- 
tain bacteria  which  have  been  introduced  with  the 
bean  develop  in  sharply  defined  horizontal  planes, 
which  slowly  ascend.  Certain  varieties  form  several 
planes  above  one  another.  I  have  had  these  interest- 
ing statements  investigated  by  Mr.  Miodowski,  who 
corroborated  them  in  great  measure.  But  instead  of 
the  non-sporulating  bacillus  perlibratus  Bey.,  which 
usually  formed  the  planes  in  Beyerinck's  experi- 
ments, we  found  chiefly  an  organism  allied  to  ba- 
cillus mesentericus  and  bacillus  subtilis  {vide  Bey- 
erinck:  C.  B.,  XIV.,  827,  and  Miodowski:  Diss., 
Wiirzburg,  1896). 

Schenk  has  observed  a  positive  thermotropism. 
If  a  hanging  drop  containing  bacteria  is  warmed  at 
one  point  with  a  warm  wire  (temperature  difference 
8°-10°)  the  bacteria  congregate  in  that  spot  (C. 
B.,  XIY.). 

2.    OPTICAL  EFFECTS. 

Phosphorescent  bacteria  are  distributed  quite 
widely,  especially  in  and  near  salty  media  (the  ocean, 
rivers,  salted  fish) ,  and  a  considerable  number — main- 
ly bacilli  and  vibriones — have  been  carefully  studied. 
Phosphorescence  is  a  vital  symptom  and  does  not 
depend  upon  the  oxidation  of  a  photogenic  substance 
secreted  by  the  bacteria  (K.  B.  Lehmann  and  ToU- 
hausen :  C.  B. ,  V. ,  785) .  It  is  destroyed  by  all  factors 
which  injure  the  life  of  the  bacteria;  cold  produces 
rigidity  of  the  organisms  and  interrupts  the  phos- 
phorescence as  long  as  it  lasts.  High  temperatures, 
acids,  chloroform,  etc.,  interfere  temporarily  with  the 
8 


114  ATLAS   OF   BACTERIOLOGY. 

phosphorescence.  Living  bacteria  can  always  be  ob- 
tained from  phosphorescent  cultures,  and  a  filtered 
culture  free  from  germs  is  always  devoid  of  phos- 
phorescence. But  although  the  organism  cannot  give 
light  without  life,  it  may  live  without  giving  light — 
for  example,  in  an  atmosphere  of  carbonic  acid.  In 
like  manner  the  muscles  cannot  contract  without  life, 
but  they  may  be  alive  without  contracting. 

According  to  Beyerinck  (C.  B.,  YIII.,  pp.  716  and 
651),  who  includes  all  phosphorescent  bacteria  in  one 
(physiological)  genus,  photobacterium,  they  require 
peptone  and  oxygen  in  order  to  produce  light.  Four 
of  his  six  varieties  also  require,  in  addition  to  pep- 
tone, a  supply  of  carbon  which  may  also  contain 
nitrogen.  Small  amounts  of  sugar  (dextrose,  levu- 
lose,  galactose,  maltose),  glycerin,  and  asparagin  act 
in  this  way.  In  some  varieties  a  higher  percentage 
of  sugar  causes  cessation  of  the  phosphorescence, 
after  the  formation  of  acids  and  marked  fermentation. 

When  the  phosphorescence  is  to  be  maintained,  we 
would  recommend  a  gelatin  nutrient  medium,  made 
by  cooking  fish  in  sea  water,  to  which  one  per  cent 
peptone,  one  per  cent  glycerin,  and  one-half  per 
cent  asparagin  have  been  added.  But  even  in  this 
medium  phosphorescence  is  soon  lost  if  inocula- 
tions are  infrequent,  so  that  in  the  majority  of  labor- 
atories the  phosphorescent  bacilli  do  not  emit  light. 
By  repeated  rapid  transfers  to  a  suitable  nutrient 
medium  we  can  often  succeed  in  restoring  the  photo- 
genic power.  I  recommend  that  two  salt  herrings  be 
cooked  in  one  litre  of  water,  and  ten  per  cent  gelatin 
added  to  the  filtrate  without  neutralization. 


THE   EFFECTS   OF  BACTERIA.  115 


3.    THERMIC  EFFECTS. 

The  development  of  heat  during  the  metabolism 
of  bacteria  is  not  noticeable  in  our  ordinary  cultures 
on  account  of  its  slight  amount.  Even  luxuriantly 
growing,  fermenting  fluid  cultures  do  not  reveal  to  the 
hand  any  noticeable  production  of  heat. 

But  there  is  no  doubt,  on  the  other  hand,  that  the 
heat  given  out  by  moist  decomposing  organic  matters, 
such  as  beds  of  tobacco,  hay,  manure,  etc.,  depends, 
at  least  in  part,  on  bacterial  activity.  In  view  of  the 
high  temperature  produced,  it  is  very  probable,  ac- 
cording to  Lydia  Kabinowitsch,  that  the  thermophilic 
bacteria  take  part  in  the  process.  Careful  investiga- 
tions concerning  the  producers  of  these  high  temper- 
atures are  still  wanting  (vide  Eabinowitsch :  Z.  H., 
XX.,  163). 

4.    CHEMICAL  EFFECTS. 

The  chemical  actions  of  bacteria,  which  are  accom- 
panied in  part  by  the  production  of  light,  and  always 
by  the  production  of  heat,  are  known  only  in  their 
main  outlines,  despite  the  extremely  numerous  and 
successful  investigations  of  the  last  twenty-five  years. 
In  many  cases  we  know  only  the  final  products,  and 
have  no  accurate  information  concerning  the  mechan- 
ism of  their  development,  the  intermediate  pro- 
ducts, and  the  substances  which  appear  in  small 
quantities. 

We  may  distinguish  the  following  three  principal 
varieties  of  chemical  efl'ects : 

1.  The  bacteria  store  up  their  cell  substance. 


116  ATLAS   OF   BACTERIOLOGY. 

2.  The  bacteria  excrete  ferments,  designed  to  make 
the  surrounding  nutrient  medium  more  suitable  for 
assimilation.  Tho  products  which  develoj^  at  this 
time  in  the  vicinity  of  the  bacteria  may  be  called 
transformation  products. 

3.  The  bacteria  assimilate  some  substances  and 
excrete  others — true  products  of  disassimilation.  A 
separation  of  fermentative  products  and  disassimila- 
tive  products,  such  as  is  still  attempted  at  times,  is 
incorrect  because  the  substances  are  only  fermented 
when  thej^  have  previously  entered  the  bacterium 
cell.  Hence  fermentation  products  are  products  of 
disassimilation  under  the  influence  of  special  nutri- 
tion (vide  page  124). 

I.  Bacterial  Ferments  and  the  Changes  Produced 
BY  Them. 

Under  the  term  ferments  in  the  narrower  sense 
(enzymes)  we  refer  to  chemical  bodies  which,  in  mini- 
mum amounts  and  without  being  used  up,  are  able  to 
separate  large  amounts  of  complicated  organic  mole- 
cules into  simple,  smaller,  more  soluble  and  diffusible 
molecules.* 

Ferments  may  be  regarded  as  chemical  only  when 
we  can  prove : 

1.  That  the  fermentation  continues  in  the  presence 
of  substances  (for  example  phenol,  three  per  cent; 
thymol,  .01  per  cent;  chloroform,  ether)  which  kill 
bacteria  but  do  not  endanger  ferments ;  or 

2.  That  the  germless  filtrate  of  the  bacterial  culture 

*  This  definition  does  not  hold  good  for  a  single  ferment,  the 
milk  ferment,  which  coagulates  the  milk  {nde  page  123) . 


THE  EFFECTS  OF  BACTBBIA.  117 

through  a  cIslj  or  porcelain  cylinder  possesses  the 
power  of  fermentation ;  or 

3.  That  this  power  inheres  in  a  sterile  preparation 
of  the  ferment,  made  in  the  shape  of  a  powder. 

Of  the  extremely  numerous  details  which  we  have 
learned  from  Fermi's  methodical  and  thorough  inves- 
tigations, we  can  here  give  only  the  most  important. 
All  ferments  dialyze  as  little  as  ordinary  albuminoids 
through  good  parchment  paper. 

Proteolytic — i.e.,  albumin-dissolving  enzymes — are 
widely  distributed.  The  liquefaction  of  the  gelatin, 
which  is  chemically  allied  to  albumin,  in  our  nutrient 
media  is  sure  evidence  of  the  presence  of  a  proteolytic 
ferment.  As  the  reaction  at  which  the  gelatin  is  dis- 
solved is  always  or  may  be  alkaline,  the  bacteria 
cultures  do  not  contain  pepsin  (which  is  effective  only 
with  acid  reaction)  but  trypsin.  The  different  bac- 
terio-trypsins  vary  greatly  in  their  resistance  to  heat 
(in  a  moist  condition  they  tolerate  a  temperature  of 
from  55°-70°  for  one  hour),  their  sensitiveness  to  dif- 
ferent acids,  etc.  Some  are  efficient  even  when  a  con- 
siderable amount  of  acid  has  been  added,  but  they 
never  act  better  than  in  an  alkaline  reaction. 

The  action  on  fibrin  is  much  weaker  than  that  on 
gelatin,  and  hence  Fermi  has  recommended  the  fol- 
lowing method  as  the  most  convenient  and  certain 
demonstration  of  the  presence  of  even  a  trace  of  pro- 
teolytic ferment.  A  non-neutralized  solution  is  made 
of  about  seven  per  cent  gelatin  in  one  per  cent  car- 
bolic acid  and  equal  amounts  are  placed  in  test  tubes 
of  the  same  size.  The  solution  to  be  tested  for  the 
ferment  is  then  placed  on  the  solid  gelatin,  after  re- 
ceiving two  per  cent  carbolic  acid.     We  can  then  read 


118  ATLAS  OF  BACTEKIOLOGY. 

off  on  a  millimetre  scale,  at  the  temperature  of  the 
room,  the  rate  at  which  the  liquefaction  of  the  gelatin 
proceeds  for  days  and  weeks.  Qualitative  tests  may 
be  simply  made  by  using  1  c.c.  of  a  liquefied  gelatin 
culture  which  has  been  sterilized  with  carbolic  acid.  * 
This  material  also  suffices  in  testing  the  influence  of 
the  nutrient  medium  upon  the  formation  of  the  fer- 
ment. By  this  method  we  may  also  compare  the 
action  of  different  degrees  of  concentration  of  differ- 
ent bacterio-trypsins.  The  less  the  percentage  of 
gelatin  and  the  nearer  the  temperature  to  incubating 
temperature,  the  more  certainly  do  we  obtain  the 
action  of  even  traces  of  ferment.  In  such  critical 
cases  the  experiment  is  continued  for  two  weeks  and 
we  then  note  whether  the  test  tubes  in  the  refrigera- 
tor, provided  with  the  ferment,  remain  fluid,  while 
the  control  tubes  remain  rigid. 

In  order  to  demonstrate  the  production  of  a  true 
peptone,  we  proceed  in  the  following  way : 

The  variety  of  bacteria  in  question  is  cultivated 
upon  a  fluid  albuminous  nutrient  medium  free  from 
peptone  (blood  serum,  milk  serum,  milk).  If  the 
culture  grows  well,  all  the  albuminoids,  with  the 
exception  of  the  peptone,  are  precipitated  by  the  ad- 
dition of  solid  ammonium  sulphate  (about  30  gm.  to 
20  c.c).  Milk  and  milk  serum  may  be  warmed  to 
60°-80°,  blood  serum  to  about  40°.  The  precipitate 
is  then  filtered,  the  filtrate  cooled;  a  part  is  made 
strongly  alkaline  by  the  addition  of  potash,  and  one- 
per-cent  solution  of  copper  sulphate  is  then  added 

*  As  a  matter  of  course  we  must  never  fail  to  make  a  control 
test  with  two-per-cent  solution  of  carbolic  acid  in  water  (free 
from  germs) . 


THE  EFFECTS  OF  BACTERIA.  119 

drop  by  drop.  The  appearance  of  a  rose  color  indi- 
cates the  presence  of  peptone.* 

The  formation  of  proteolytic  ferments  varies  in 
many,  perhaps  in  all,  species  to  a  much  greater  extent 
than  we  would  imagine  from  the  ordinary  descrip- 
tions. In  the  case  of  two  phosphorescent  vibriones 
Beyerinck  found  that  one  which  at  first  liquefied 
gelatin  very  slowly,  did  so  more  rapidly  after  longer 
culture,  while  the  other  variety  acted  in  the  opposite 
way.  Katz  made  a  similar  observation  in  experi- 
ments on  Australian  phosphorescent  bacteria.  Max 
Gruber  and  Firtsch  have  watched  very  closely  the 
development  of  feebly  liquefying  races  in  vibrio  pro- 
teus  (A.  H.,  VIII.,  369),  and  similar  statements  have 
been  made  concerning  cholera  vibrio,  bacterium 
vulgare,  and  micrococcus  pyogenes.  Indeed,  some 
observers  have  even  seen  a  liquefying  streptococcus 
pyogenes. 

We  have  also  observed  in  many  varieties  that  on 
thin  plates  the  individual  distinctly  visible,  super- 
ficial colonies  exhibit  very  different  degrees  of  lique- 
faction. In  fact  a  beginner  would  be  convinced  that 
he  had  to  deal  with  several  varieties. 

It  is  to  be  regretted  that,  as  a  result  of  these  obser- 
vations, one  of  the  most  convenient  diagnostic  aids, 
viz.,  the  liquefaction  of  gelatin,  has  lost  consider- 
ably in  value. 

The  causes  of  the  increase  and  decrease  of  liquefac- 
tion with  prolonged  culture  are  looked  for  in  our 
artificial  nutrient  media,  or  in  the  influence  of  the 

*  Recent  investigations  have  shown,  however,  that  in  addition 
to  peptone  a  few  albumoses  remain  unprecipitated  in  part  by 
ammonium  sulphate. 


120  ATLAS   OF   BACTEEIOLOGY. 

products  of    disassimilation  of  tlie  micro-organism, 
but  we  are  unable  to  give  any  positive  data. 

Concerning  the  influence  of  the  nutrient  media  upon 
the  formation  of  trypsin  in  a  culture  or  the  liquefac- 
tion of  the  gelatin,  the  following  facts  are  known: 

1.  The  majority  of  circumstances  which  impair  the 
growth  of  a  variety  of  bacteria  upon  a  nutrient 
medium  also  interfere  with  liquefaction — for  exam- 
ple, the  addition  of  phenol,  or  a  large  percentage  of 
glycerin.  Wood  found  that  the  impaired  power  of 
liquefying  gelatin,  which  was  produced  by  phenol, 
was  transmitted  during  several  generations  upon  a 
good  nutrient  medium  (C.  B.,  YIII.,  266). 

2.  The  liquefying  facultative  anaerobics  do  not 
liquefy  gelatin*  in  hydrogen  and  nitrogen,  but  they 
do  in  carbonic  acid,  if  they  are  able  to  grow  in  the 
latter  medium.  As  the  gases,  according  to  Fermi, 
have  no  effect  upon  the  action  of  the  ferment,  they 
must  influence  the  formation  of  the  ferment.  Strict 
anaerobics,  on  the  other  hand,  produce  the  most  pro- 
nounced liquefaction  of  gelatin. 

3.  In  many  bacteria  the  addition  of  sugar  inter- 
feres not  with  their  growth,  but  with  the  liquefaction 
of  gelatin — for  example,  in  bacterium  vulgare  (proteus 
vulgaris)  but  not  in  bacillus  subtilis  (Kuhn:  A.  H., 
Xin.,  70).  This  is  explained,  perhaps,  by  the  fact 
that  bacterium  vulgare  produces  an  acid  from  sugar, 
and  the  vulgare  trypsin  is  very  sensitive  to  acids. 
Upon  10  c.c.  of  a  one-per-cent  grape-sugar  gelatin,  in 
five  days  bacterium  vulgare  produced  3.7  c.c.  of  one- 
tenth  normal  acid,  vibrio  proteus  2.1   c.c,    bacillus 

*With  the  single  exception  of  bacterium  prodigiosum,  but 
this  also  ceases  to  liquefy  on  the  addition  of  grape  sugar. 


THE   EFFECTS  OF   BACTERIA.  121 

subtilis  1.7  c.c,  bacillus  anthracis  0.9  c.c. ;  bacte- 
rium vulgare  was  the  only  one  which  did  not  produce 
liquefaction. 

4.  In  fluid,  non-albuminous,  glycerin-containing 
(free  from  sugar)  nutrient  media,  very  few  bacteria 
produce  proteolytic  ferments — for  example,  bacterium 
prodigiosum  and  bacterium  pyocyaneum.  The  pro- 
duction of  ferment  also  apj^ears  to  be  less  on  pep- 
tone bouillon  than  on  peptone  -  bouillon  gelatin 
(Fermi). 

Upon  albuminous  nutrient  media  the  liquefying 
bacteria  produce  bitter  products  of  disassimilation 
—for  example,  this  is  done  in  milk  by  very  many 
varieties  (Hiippe)  An  enumeration  of  the  trypsin- 
forming  varieties  is  unnecessary  because  they  are 
characterized  as  trypsin-producers  by  their  liquefac- 
tion of  gelatin. 

The  other  ferments  have  been  studied  less  care- 
fully. 

Diastatic  ferments  convert  starch  into  sugar.  They 
are  demonstrated  in  the  following  manner:  A  thin 
starch  paste  containing  one  per  cent  thymol  is  com- 
bined with  a  culture  to  which  one  to  two  per  cent  thy- 
mol has  been  added,  and  is  kept  six  to  eight  hours  in 
the  incubating  chamber.  A  little  Fehling's  solution  is 
then  added  and  sugar  is  recognized  by  the  reduction 
of  copper  (reddish-yellow  precipitate)  on  boiling. 
We  can  also  make  a  direct  examination  of  mashed 
potato  cultures  of  the  bacteria  by  boiling  the  cultures 
and  testing  the  extract. 

According  to  Fermi  about  one-third  of  the  varie- 
ties examined — only  upon  albuminous  nutrient  media 
— possess  the  power  of  forming  such  a  ferment  (A.  H., 


122  ATLAS   OF   BACTERIOLOGY. 

X.,  and  C.  B.,  XII.,  p.  713)  viz.,  the  bacilli  of  the  sub- 
tilis  group  (anthrax,  megatherium,  Fitzianus,  etc.), 
the  vibriones  related  to  the  cholera  vibrio,  also  micro- 
coccus tetragenus,  micrococcus  mastitidis,  bacterium 
violaceum,  bacterium  mallei,  bacterium  pyogenes 
foetidum,  bacterium  phosphorescens,  bacterium  pneu- 
moniae, bacterium  synxanthum,  bacterium  aceticum; 
the  others  are  not  active  or  are  doubtful.  In  addition 
all  the  actinomyces  and  oospora  varieties  (with  the  ex- 
ception of  oospora  carnea).  The  majority  of  the 
varieties  mentioned  subsequently  convert  the  sugar 
into  acid  but  some  do  not,  for  example,  bacillus 
subtilis. 

Inverting  ferments  (i.e.,  those  which  convert  cane 
sugar  into  grape  sugar)  are  rare,  according  to  Fermi 
and  Montesano.  They  are  demonstrated  in  the  fol- 
lowing way :  A  one  to  two  per  cent  solution  of  cane 
sugar  containing  one  per  cent  of  carbolic  acid  is 
added  to  a  culture  containing  one  per  cent  of  carbolic 
acid.  After  a  few  hours  we  test  whether  the  fluid 
reduces  Fehling's  solution;  as  is  well  known,  cane 
sugar  does  not  produce  this  reaction.  Control  tests 
with  a  solution  of  cane  sugar  alone  are  always  neces- 
sary. Bacteria  invertin  tolerates  (always?)  a  tem- 
perature of  100°  for  more  than  an  hour,  and  also  de- 
velops upon  a  non-albuminous  nutrient  medium  if 
glycerin  is  present.  The  above-named  writers  men- 
tion only  the  following  forms  as  producers  of  invert- 
ing ferments ;  bacillus  megatherium,  bacillus  kiliense, 
bacillus  fluorescens  liquefaciens,  bacterium  vulgare, 
vibrio  cholerse  and  Metschnikovii. 

The  attempts  to  find  a  ferment  similar  to  emulsin 
have    been    unsuccessful.       Micrococcus    pyogenes 


THE   EFFECTS   OF   BACTERIA.  123 

tenuis  transfori][is  amygdalin  into  benzaldehyd,  but 
this  function  cannot  be  separated  from  cell  life. 

Eennet  ferments — i.e.,  bodies  which  coagulate  milk 
of  a  neutral  (or  amphoteric)  reaction  and  indepen- 
dently of  the  action  of  acids — are  not  wanting  among 
the  bacteria.  It  can  be  demonstrated,  for  example, 
in  not  too  old  cultures  of  bacterium  prodigiosum 
which,  sterilized  at  55°-60°,  can  easily  coagulate 
sterilized  milk  solid  in  one  or  more  days  (Gorini :  C. 
B.,  XIL,  666). 

So  far  as  I  know,  thorough  investigations  concern- 
ing the  distribution  of  this  ferment  are  still  lacking. 
We  may  suspect  it  in  all  varieties  which  coagulate 
milk  without  possessing  the  power  of  forming  lactic 
acid  out  of  milk  sugar. 

II.  The  Chemical  Actions  of  Bacterial 
Metabolism. 

Like  the  production  of  ferments,  the  majority  of 
the  other  chemical  actions  of  bacteria  depend,  in 
great  measure,  on  the  nutrient  medium.  This  is 
most  striking  when  the  growth  of  many  forms  of 
bacteria  is  observed  upon  an  albuminous  nutrient 
medium,  which  at  one  time  is  free  from  sugar,  at 
another  time  contains  sugar.  In  the  former  event, 
apart  from  pigment  substances  and  perhaps  some 
badly  smelling  substances,  hardly  any  perceptible 
metabolic  products  are  formed ,  but  in  the  latter  event 
there  is  often  a  very  striking  change,  characterized 
by  the  development  of  gas  and  active  production  of 
acid.  The  organism  joroduces  fermentation  in  the 
sugar-containing  medium,  in  the  other  it  does  not. 


124  ATLAS   OF   BACTERIOLOGY. 

On  account  of  the  practical  (and  diagnostic)  im- 
portance of  the  fermenting  power  we  must  here  give 
a  precise  definition  of  this  process.  The  term  fermen- 
tation is  used  in  literature  in  various  senses. 

1.  Some  writers  call  every  typical  decomposition 
produced  by  bacteria  a  fermentation,  and  speak,  for 
example,  of  the  putrid  fermentation  of  albuminoids. 

2.  Others  confine  the  term  fermentation  to  proc- 
esses which  are  attended  with  the  visible  develop- 
ment of  bubbles  of  gas.  According  to  this  definition 
the  conversion  of  nitric  acid  into  nitrogen  is  a  fer- 
mentation as  well  as  the  fermentation  of  milk  sugar 
by  bacterium  acidi  lactici. 

3.  Still  others  use  the  term  only  in  cases  of  decom- 
position of  hydrocarbons  with  or  without  the  forma- 
tion of  gas. 

It  seems  to  me  that  the  term  fermentation  is  always 
in  place  when  it  can  be  shown  that  an  organism,  in 
addition  to  or  instead  of  its  other  metabolic  products, 
forms  one  or  a  few  special  metabolic  products  in  an 
unusual  amount — metabolic  products  which  are  al- 
most always  derived  from  the  merely  superficial 
splitting  up  of  a  bacterial  nutrient  which  is  easily  split 
up.  Oxidative  fermentation  is  rarer.  A  necessary 
condition  of  fermentation  is  the  presence  of  a  definite 
nutrient  matter  which  the  bacteria  attack  with  special 
ease,  often  discarding  substances  which  are  less  acces- 
sible but  which  they  decompose  in  the  absence  of 
the  fermenting  substance. 

Every  fermentation  is  intended  to  carry  a  supply 
of  energy  to  the  fermenting  organism.  In  the  fer- 
mentation which  splits  up  organic  material,  this  is 
due  to  the  fact  that  the  complicated,   fermentible 


THE    EFFECTS   OF   BACTERIA.  125 

molecule  in  the  bacterial  cell  is  decomposed  into 
smaller  particles,  during  which  process  heat  is  given 
off.  I  will  illustrate  this  by  the  ordinary  form  of 
fermentation  of  sugar  in  which  the  process  is  very 
simple. 

CeHiaOe         =  SCsHeO     +  2C0, 

1  grape  sugar  =  3  alcohol  +  2  carbonic  acid. 


Or, 
Or, 


CeHisOs       =      2C3H6O3 

1  grape  sugar  =  2  lactic  acid. 

CeHiaOe       =      3C2H4O2 

1  grape  sugar  =  3  acetic  acid. 


The  organism  requires  such  a  source  of  energy, 
particularly  when  it  grows  in  the  absence  of  oxygen, 
and  there  is  a  failure  of  the  source  of  energy  at  the 
command  of  the  aerobic  varieties  and  which  consists 
in  the  oxidation  of  absorbed  substances  by  the  oxy- 
gen which  has  been  taken  up.  Hence  all  anaerobic 
varieties  are  provided  with  great  power  of  fermenta- 
tion of  sugar,  and  some  facultative  anaerobics  only 
give  rise  to  fermentation  of  a  saccharine  nutrient 
when  oxygen  is  excluded. 

In  contradistinction  to  fermentation  by  the  split- 
ting-up  process  is  the  much  rarer  oxidative  fermen- 
tation, the  best  example  of  which  is  the  production 
of  acetic  acid  from  alcohol.  Here  we  find  a  one-sided 
metabolic  activity  of  the  acetic  acid  bacteria.  These 
obtain  a  considerable  supply  of  energy,  not  by  split- 
ting up,  but  by  oxidation  of  the  absorbed  alcohol. 
The  gain  in  energy  occurs  simply  from  a  one-sided 
intensification  of  the  ordinary  nutritive  processes  of 
bacteria. 

It  is  evident  from  these  remarks  that  products  of 


126  ATLAS   OF   BACTERIOLOGY. 

fermentation  are  products  of  metabolism  like  all  the 
other  products  of  the  bacterial  cell,  and  hence  a  di- 
vision of  fermentations  in  principle  is  not  warranted. 
But  it  will  be  advisable  to  discuss  the  individual  bac- 
terial products  according  to  their  development  upon 
a  saccharine  or  non-saccharine  nutrient  medium,  and 
then  to  add  some  functions  of  the  bacteria  which  are 
manifested  by  decomposition  of  salts  of  the  fatty 
acids,  alcohols,  etc. 

A.  Functions  upon  which  the  Amount  of 
Sugar  in  the  Nutrient  Medium  Exerts 
no  Great  Influence. 

1.  Formation  of  Pigment. 

The  chemistry  of  the  pigment  matters  has  been 
very  little  studied,  but  in  recent  times  a  preliminary 
survey  has  been  made  by  some  of  Migula's  pupils. 
In  regard  to  the  fluorescent  pigments  I  follow  the 
statements  of  K.  Thumm  ( "  Arbeiten  d.  bact.  Instituts 
Karlsruhe,"  published  by  Klein  and  Migula,  Vol.  I., 
Pt.  3,  p.  291)  and  those  of  Paul  Schneider  (eod.  loco, 
Yol.  I.,  Pt.  2,  p.  201)  in  regard  to  the  other  pigments. 

1.  Ked  and  Yellow  Pigments.  According  to 
Schneider  the  twenty -seven  yellow  and  red  bacte- 
ria furnish,  in  almost  all  cases,*  pigments  which  are 
soluble  in  alcohol,  insoluble  in  water,  f  and  are  also 

*  The  coloring  matter  of  micrococcus  cereus  flavus  Passet  was 
soluble  only  in  dilute  caustic  potash. 

f  A  striking  contrast  to  these  results  is  furnished  by  M.  Freund 
(C.  f.  B.,  xvi.,  640).  In  examining  four  newly  discovered  red 
and  yellow  bacteria  he  found  the  pigment  always  soluble  in 
water,  and  insoluble  in  alcohol  and  ether. 


SUGAR  IN"  THE   NUTRIENT   MEDIUM.  127 

soluble  in  ether,  carbon  bisulphide,  benzol,  and  chlo- 
roform. 

The  large  majority,*  in  the  dry  condition,  are 
colored  bluish-green  by  concentrated  sulphuric  acid 
and  red  or  orange  by  caustic  potash,  or  they  retain 
these  colors  when  so  treated.  But  the  various  pig- 
ments show  various  chemical  differences  and  quite  a 
different  reaction  in  the  spectrum.  The  majority 
may  be  placed  unhesitatingly  in  the  large  group  of 
lipochromata  which  are  widely  distributed  in  the 
animal  and  vegetable  kingdoms,  and  to  which  belong 
the  coloring  matter  of  fat,  yolk  of  the  egg,  the  carotin 
of  carrots,  and  many  others. 

Entirely  different  from  these  substances  are  the 
pigments  of  bacterium  prodigiosum  and  bacterium 
kiliense.  These  take  a  brownish-red  color  with  con- 
centrated sulphuric  acid,  and  a  yellowish-brown 
and  yellowish-red  color  with  caustic  potash.  They 
are  allied  to  one  another  but  still  quite  distinct,  f  It 
has  often  been  assumed,  especially  on  account  of  the 
golden  shimmer  of  the  prodigiosum  culture,  that  we 
have  to  deal  here  with  a  coloring  matter  resembling 
fuchsin,  but  on  careful  examination  the  resemblance 
is  found  to  be  very  superficial. 

Violet  Pigments.     Bacterium   violaceum  and  bac- 

*  Thirteen  red  and  fourteen  yellow  bacteria  were  examined, 
and  the  only  exceptions  were  bacterium  prodigiosum  and  bacte- 
rium kiliense.  Schneider  furnishes  full  tabulated  statements 
concerning  the  reactions  of  the  alcoholic  solution  and  of  the  dry 
coloring  matter  with  various  agents,  and  also  concerning  the 
spectrum  reactions. 

f  The  fact  that  this  coloring  matter  or  one  of  its  derivatives  is 
not  entirely  insoluble  in  water  is  evident  from  the  fact  that  in 
old  agar  cultures  garnet-red  pigment  is  diffused  in  the  agar. 


128  ATLAS   OF   BACTERIOLOGY. 

terium  janthinum  were  found  to  contain  a  violet 
coloring  matter,  which  was  insoluble  in  water,  readily 
soluble  in  alcohol,  but  insoluble  in  ether,  benzol,  and 
chloroform.  In  the  dry  state  it  is  turned  yellow  hj 
concentrated  sulphuric  acid  and  emerald  green  by 
caustic  potash.  In  alcoholic  solution  it  assumes  a 
greenish  to  bluish-green  color  on  the  addition  of 
strong  acids  and  ammonia.  The  pigment  loses  its 
color  on  the  addition  of  zinc  and  sulphuric  acid. 

Claessen  and  Schneider  examined,  in  a  very  imper- 
fect manner,  the  beautiful  blue  coloring  matter  of 
bacterium  indigonaceum  Claessen.  This  pigment  is 
insoluble  in  the  ordinary  solvents;  in  hydrochloric 
acid  it  gives  at  first  a  blue,  then  a  yellowish-brown 
solution.  Other  acids  dissolve  it  but  cause  decom- 
position.    Caustic  potash  gives  a  bluish-green  color. 

Distinct  from  these  blue  coloring  matters  is  the 
blue  pigment  formed  by  bacterium  syncyaneum  (blue 
milk)  in  addition  to  bacterio-fluorescein  (vide  below) 
and  for  which  I  propose  the  term  syncyanin.  Thumm 
describes  the  pigment  as  very  unstable.  Acids  color 
it  steel  blue ;  in  slight  acidity  it  is  bluish-black,  neu- 
tral black,  and  alkaline  brownish-black. 

According  to  the  recent  investigations  of  Thumm 
the  fluorescent  pigments,  which  are  found  in  numer- 
ous cultures,  are  all  identical.  The  coloring  matter, 
for  which  I  propose  the  term  bacterio-fluorescein,  is 
lemon  yellow  and  amorphous  in  the  dry  state,  soluble 
in  water  and  dilute  alcohol,  and  insoluble  in  strong 
alcohol,  ether,  and  carbon  bisulphide.  The  watery 
solution,  when  concentrated,  has  an  orange  color, 
when  diluted,  a  pale  yellow  color;  with  acid  reaction 
it  shows   no  fluorescence,   with  neutral    reaction  a 


SUGAR   IN  THE   NUTRIENT   MEDIUM.  129 

bluish,  with  alkaline  a  green  fluorescence.  The 
fluorescence  of  the  cultures  is  at  first  blue,  later 
green,  on  account  of  the  increase  of  the  ammonia 
formed  by  the  bacteria.  The  pigment  is  not  sensi- 
tive to  oxidizing  substances.  Colorless  preliminary 
stages  have  not  been  observed.  Phosphoric  acid  and 
magnesium  are  necessary  to  the  development  of  bac- 
terio-fluorescein. 

The  variations  in  the  chromogenic  functions  have 
been  the  subject  of  numerous  investigations.  All 
possible  factors  which  have  an  unfavorable  influence 
on  the  growth  of  the  bacteria  also  diminish  the  de- 
velopment of  pigment.  After  continued  culture  upon 
unsuitable  nutrient  media  or  at  improper  tempera- 
tures, etc. ,  the  formation  of  pigment  by  later  genera- 
tions may  remain  permanently  diminished. 

For  example,  there  are  races  of  bacterium  syncy- 
aneum  which  form  no  trace  of  coloring  matter  in  agar 
or  milk,  but  on  potato  give  a  dark  color  even  to  the 
parts  around  the  culture.  The  development  of  pig- 
ment appears  to  have  been  lost  here  simply  on  account 
of  the  rare  inoculation  of  the  agar  cultures. 

At  37°  bacterium  prodigiosum  forms  no  pigment, 
and  if  the  cultures  are  kept  up  at  this  temperature  for 
a  long  time,  the  production  of  pigment  will  be  lost 
for  many  generations  even  under  favorable  conditions 
(Schottelius). 

Very  interesting  communications  are  scattered 
throughout  the  literature  on  pigment-forming  races 
among  otherwise  colorless  varieties.  For  example, 
Fawitzky  reports  yellow  to  rusty  red  colonies  of 
streptococcus  lanceolatus;  Kruse  and  Pasquale  ob- 
served colored  races  of  streptococcus  pyogenes 
9 


130  ATLAS   OF   BACTERIOLOGY. 

(Ziegler's  "Beitrage,"  XII.).  Kutscher  lias  recently 
published  the  experience  that  a  pseudo-glanders 
bacillus,  taken  from  the  animal,  had  a  bright  orange- 
red  color  only  in  the  first  culture  upon  serum,  but 
this  color  changed  to  white  after  a  few  inoculations. 
Perhaps  still  greater  importance  attaches  to  the 
often  made  observation  that,  as  the  result  of  in- 
ternal causes,  colored  and  uncolored  colonies  of  one 
variety,  for  example,  bacterium  kiliense,  occasion- 
ally develop  upon  plate  cultures. 

2.   The  Formation  of  Alkaline  Metabolic  Products  and 
Urea  Fermentation. 

According  to  v.  Sommaruga  (Z.  H.,  XII.,  273) 
aerobic  bacteria,  when  growing  in  a  non-saccharine 
nutrient  medium,  always  produce  an  alkali  from  the 
albuminoids. 

When  sugar  is  present  the  majority  of  varieties 
form  acid  out  of  the  sugar,  in  addition  to  the  produc- 
tion of  alkali,  and  the  originally  neutral  or  feebly 
acid  reaction  of  many  young  bacterial  cultures  is  ex- 
plained simply  by  a  slight  percentage  of  sugar  in  the 
bouillon  (derived  from  the  meat).  When  the  sugar 
is  used  up,  the  production  of  alkali  becomes  more 
pronounced  (Th.  Smith). 

So  far  as  we  know  at  present,  the  alkaline  bodies 
produced  are  ammonia  (occasionally  perceptible  to 
the  sense  of  smell),  amine  and  ammonia  bases.  In 
order  to  determine  the  degree  of  production  of  the 
alkali,  we  titrate  tubes  which  contain  10  c.c.  peptone 
bouillon,  uninoculated,  and  one  to  eight  days  after 
inoculation  with  one-tenth  normal  acid  and  phenol- 


SUGAR   IlSr   THE   NUTRIENT   MEDIUM.  131 

plithalein  as  indicator.     The  difference  in  the  titra- 
tions gives  the  increase  of  alkali. 

The  following  will  serve  as  an  illustration  of  the 
production  of  alkali  by  bacteria  which  in  the  pres- 
ence of  sugar  form  acid  actively  (5-7  c.c.  normal  acid 
to  100  c.c).  One  hundred  cubic  centimetres  of  a 
nutrient  medium  containing  traces  of  meat  sugar  and 
rendered  neutral  by  phenolphthalein  used  up : 

When  Inoculated  with  Bacterium  Coli. 

At  the  end  of  five  days 0.1    normal  sodium. 

At  the  end  of  ten  days 0.1    normal  sodium. 

At  the  end  of  fifteen  days  0.25  normal  acid. 

A  special  form  of  alkali  production  by  bacteria  is 
the  conversion  of  urea  into  ammonium  carbonate: 
CO(NH,),  +  2H,0  =  C03(NH,),. 

Leube  (Yirch.  Arch.,  100,  p.  540)  first  isolated  a 
few  organisms  frona  decomposing  urine  which  pro- 
duced ammonia  from  urea :  micrococcus  urese  Leube, 
bacillus  ure?e  Leube.  This  is  also  done  by  sarcina  pul- 
monum  and  a  few  other  unnamed  varieties.  Fliigge 
has  described  a  micrococcus  ureae  liquefaciens. 

We  have  cultivated  a  large  number  of  white  lique- 
fying and  non-liquefying  cocci  and  rods  from  decom- 
posing urine.  None  of  them  possessed  in  any  strik- 
ing degree  the  power  of  setting  free  ammonia  from 
diluted  urine  or  a  nutrient  medium  treated  with  urea, 
although  they  flourished  in  these  solutions.  It  can- 
not be  denied  that  natural  urea  fermentation  depends 
partly  on  symbiosis. 

The  ability  to  decompose  urea  does  not  seem  to  be 
very  widespread.  Among  twenty-four  organisms  ex- 
amined Warington  found  that  two  alone  (micrococcus 


132  ATLAS    OF    BACTERIOLOGY. 

ureae  and  bacillus  fluorescens)  produced  ammoniacal 
decomposition  of  urine. 

Among  sixty  varieties  only  three  (bacterium  vul- 
gare,  bacterium  prodigiosum,  and  bacterium  kiliense) 
developed  a  distinct  ammoniacal  odor  in  sterilized 
human  urine. 

Leube  employed  Jacksch's  nutrient  solution :  In  1 
litre  0.125  acid  potassium  phosphate,  0.062  mag- 
nesium sulphate,  5  gm.  Seignette  salts,  which  were 
sterilized .  by  boiling.  To  the  sterile  solution  he 
added  3  gm.  urea  which  had  been  sterilized  in  a  dry 
state  at  106^  (boiling  of  urea  solutions  is  to  be 
avoided  because  a  part  of  the  urea  is  thus  converted 
into  ammonium  carbonate) .  In  order  to  demonstrate 
the  presence  of  the  ammonia  Leube  employed  Ness- 
ler's  reagent,  a  very  sensitive  test.  For  the  study 
of  the  quantitative  relations  vide  Brodmeier  (C.  B., 
XVIII.,  p.  380).  Urea  is  not  decomposed  upon  a 
nutrient  medium  which  contains  sugar.  Burri,  Her- 
feldt,  and  Stutzer  (C.  B.,  Pt.  II.,  Vol.  I.,  284)  recently 
described  three  rods  which  decompose  urea  very 
vigorously. 

In  addition  to  ammonia  Brieger's  investigations 
have  disclosed  a  large  number  of  basic  crystalline 
nitrogenous  bodies  as  products  of  bacterial  metab- 
olism. These  bodies  are  now  usually  called  pto- 
mains  (7rrw//a,  putrefaction)  or  putrefaction  alkaloids, 
when  they  are  not  poisonous,  and  toxins*  when  they 
are  poisonous. 

*  With  the  growth  of  our  knowledge  of  bacterial  poisons  the 
conception  of  toxins  has  been  enlarged,  so  that  now  the  major- 
ity of  writers  call  all  bacterial  poisons  toxins,  irrespective  of 
their  chemical  constitution. 


SUGAR  IK  THE   NUTKIENT  MEDIUM.  133 

So  far  as  they  have  been  closely  examined,  the 
majority  belong  to  the  following  groups : 

1.  Amins.     Methylamin,    dimethylamin,   and   tri- 

/CH3  /CH3 

N-H  N— CH3 

\H  \H 

methylamin,  similar  to  ethylamin,  diethylamin,  and 

/CH3 

N— CH3 

xCHs 

triethylamin.      Ethylendiamin  ||    „     and  its  homo- 

logues,  dimethylethylendiamin-putrescin,  with  which 
sepsin  is  isomeric;  pentamethylendiamin  is  called 
cadaverin.     The  most  virulent  one  is  ethylendiamin. 

2.  Ammonium  Bases.     The  best  known  is  cholin- 

/CH3 

^CH3 
bilineurin  =  N^- — CH3 

Muscarin  (CgHj^NOg)  is  closely  allied,  and  likewise 
vinylcholin  (C.H.^NO)  and  neuridin  (C,H,,NJ. 

3.  Pyridin  derivatives.  Derived  from  pyridin 
CgH.N ;  the  principal  ones  that  have  been  found  are 
coUidin  C^H^N  and  i^arvolin  CgHjgN. 

4.  Indol  (C«H,N)  and  skatol  (C,H,N),  vide  page  142. 

In  addition,  amido  acids  (leucin,  tyrosin,  etc.),  sub- 
stances allied  to  guanidin  (C(NH)(NH2)2)  and  numer- 
ous other  imperfectly  characterized  bodies  have  been 
discovered.  It  would  be  useless  to  mention  them 
here  as  the  poisonous  ones  are  no  longer  regarded  as 
the  true  viruses  of  the  disease  {vide  page  135) . 


134  ATLAS   OF   BACTERIOLOGY. 

The  isolation  of  these  bodies  can  only  be  hinted  at. 
According  to  Brieger's  method,  which  is  usually 
employed,  the  culture  of  a  feebly  acid  reaction  (hy- 
drochloric acid)  is  brought  to  a  boil  for  a  short  time, 
the  filtrate  then  condensed  into  a  syrup,  dissolved  in 
ninety-six-per-cent  alcohol,  and  then  freed  from  im- 
purities (especially  traces  of  albumin)  by  alcoholic 
lead  acetate.  The  lead  is  then  removed,  the  filtrate 
concentrated,  and  from  this  the  mercurial  binary 
compound  of  the  ptomains  are  precipitated  with  al- 
coholic solution  of  corrosive  sublimate.  When  the 
alcohol  has  been  removed  by  heat  and  the  mercury 
by  sulphuretted  hydrogen,  the  characteristic  gold  and 
platinum  binary  compounds  are  produced,  or  we  at- 
tempt directly  to  obtain  the  crystalline  chlorhydrates 
and,  by  the  aid  of  caustic  soda,  the  free,  often  fluid. 


Some  ptomains,  like  very  many  vegetable  alka- 
loids, can  be  easily  obtained  with  ether  in  a  watery 
solution  as  soon  as  they  have  been  set  free  by  potash 
lye.  But  Brieger's  method  is  much  better  because  it 
secures  many  substances  which  do  not  dissolve  in 
ether. 

3.  Formation  of   Complicated   "Albumin-like''    Toxic 
Metabolic  Products, 

("  Toxalbumins, "  Toxins.) 

In  connection  with  the  discussion  of  the  relatively 
simple,  basic,  more  or  less  poisonous  metabolic  prod- 
ucts of  bacteria,  we  may  make  a  few  brief  remarks 
on  other  bacterial  poisons.  In  the  present  state  of 
our  knowledge  they  may  be  divided  into  two  classes. 


SUGAR   IK   THE   NUTRIENT   MEDIUM.  135 

1.  Bacterial  Proteins  (Buchner). — This  term  refers 
to  certain  albuminoid  substances  which  produce  fe- 
ver (pyogenic)  and  inflammation  (phlogogenic). 
They  are  obtained  by  boiling,  for  several  hours,  po- 
tato cultures  together  with  one-half-per-cent  potash 
lye  (about  fifty  volumes  potash  to  one  volume  bac- 
terial substance).  The  clear  fluid,  filtered  through 
paper,  allows  the  precipitation  of  the  proteins  after 
careful  feeble  acidulation.  The  proteins  may  then 
be  washed,  dried,  and,  before  using,  dissolved  in  a 
weak  solution  of  soda. 

The  best-known  protein  is  Koch's  tuberculin.  Mal- 
lein  also  belongs  to  this  category.  According  to 
Buchner  and  Roemer  all  bacterial  proteins  have  es- 
sentially the  same  action.  According  to  Mathes  deu- 
teroalbumose,  which  is  obtained  by  the  action  of 
pepsin  on  albumin  and  has  no  connection  whatever 
with  bacteria,  produces  the  same  effects  on  tubercu- 
lous guinea-pigs. 

2.  '^  Toxalbumins.''  —  C.  Fraenkel  and  Brieger 
(Deut.  med.  Wschr.,  1890,  4  and  5)  confirmed  in  great 
measure  the  statements  of  earlier  observers  (Christ- 
mas, Roux  and  Yersin,  Hankin)  that  measures  which 
precipitate  albumin  will  also  precipitate  from  the 
bouillon  cultures  of  many  bacteria  amorphous  poisons 
which  exert  an  intense  and  usually  specific  (similar 
to  the  living  culture)  toxic  action.  They  called  these 
poisons  toxalbumins  and  considered  them  analogous 
to  the  toxic  albuminoid  bodies  in  many  plants  (ricin 
in  ricinus  communis,  abrin  in  abrus  precatorius, 
etc.).  The  majority  of  investigators  regarded — and 
some  still  regard — these  poisons  as  "  labile"  albumi- 
noids,  which  are    derived  from  the   bacterial  cell. 


136  ATLAS   OF   BACTERIOLOGY. 

They  are  also  regarded  as  analogous  to  snake  poisons 
and  to  the  enzymes.  With  these  bodies  they  share  a 
great  sensitiveness  to  heat,  reagents,  light,  etc. 

The  toxalbumins  are  obtained  as  a  raw  product  by 
precipitating,  with  absolute  alcohol  or  ammonium 
sulphate,  old  bouillon  cultures  of  the  bacteria  which 
have  been  concentrated  in  a  vacuum,  and  which  have 
been  freed  from  living  germs  by  passing  through  a 
porcelain  filter.  If  the  ammonia  salt  has  been  used, 
this  is  removed  from  the  filtered  precipitate  by  dialy- 
sis with  flowing  water  in  a  parchment  coil  and,  after 
renewed  concentration  in  a  vacuum,  precipitation  of 
the  bodies  with  absolute  alcohol.  It  has  recently 
been  discovered  that  zinc  chloride  precipitates  these 
bodies  quantitatively,  and  the  toxins  can  be  separated 
from  the  precipitate  by  the  aid  of  sodium  phos- 
phate (Brieger  and  Boer:  Z.  H.,  XXI.,  268). 

From  the  beginning,  however,  doubts  were  ex- 
pressed whether  these  toxalbumins  were  not  merely 
carried  down  by  the  precipitated  albumin  and  perhaps 
had  no  connection  with  the  albumin. 

In  the  case  of  tetanus  poison,  Brieger  and  Cohn 
(Z.  H.,  XY.,  1)  succeeded  in  obtaining  from  the  raw 
product,  by  means  of  lead  acetate  and  ammonia,  a 
pure  virus  which  showed  a  faint  violet  color  with 
copper  sulphate  and  soda  lye  but  gave  no  albumin 
reaction ;  it  is  free  from  phosphorus  and  almost  en- 
tirely from  sulphur.  It  thus  seems  to  be  proven  that 
the  tetanus  virus  is  not  an  albuminoid. 

The  statements  of  Uschinsky  that  he  obtained  an 
albuminoid  tetanus  virus  and  diphtheria  virus  upon 
a  non-albuminous  nutrient  medium  have  not  been 
tested  hitherto  because  German   observers  did  not 


SUGAR  INT   THE   NUTRIENT   MEDIUM.  137 

succeed  in  securing  a  sufficient  growth  of  these  organ- 
isms upon  a  non-albuminous  medium.  Brieger  and 
Cohn  found  that  cholera  vibriones  formed  a  non- 
albuminous  virus  upon  the  Uschinsky  nutrient  me- 
dium. The  diphtheria  virus  is  also  recognized  now 
as  free  from- albumin  (Brieger  and  Boer:  L  c). 

It  is  becoming  more  and  more  customary  to  call 
the  bacterial  poisons  simply  toxins  and  to  ignore 
entirely  the  existence  of  the  above-described  crystal- 
lizable  toxins  of  simple  constitution. 

Concerning  the  other  characteristics  of  these  tox- 
ins I  will  make  a  few  remarks,  taking  the  tetanus 
virus  as  an  illustration  (Brieger  and  Cohn:  I.  c). 
Waterjt  solutions  are  not  coagulated  by  heat  but  lose 
their  poisonous  properties  in  time.  The  addition  of 
small  amounts  of  acid  or  alkali  to  produce  solution, 
and  prolonged  transmission  of  carbonic  acid  and 
sulphuretted  hydrogen  impair  the  toxicity  very  ma- 
terially. In  the  dry  state  the  virus  tolerates  a  tem- 
perature of  70°  very  well  for  a  long  time,  higher 
temperatures  decompose  it  rapidly.  When  dried 
and  protected  from  light,  air,  and  moisture,  it  is 
converted  slowly  into  an  inert  substance.  It  is  bet- 
ter preserved  when  covered  with  absolute  alcohol, 
anhydrous  ether,  and  the  like. 

The  virulence  of  the  purest  tetanus  virus  is  almost 
inconceivable.  A  mouse  weighing  15  gm.  is  killed 
by  0.00005  mgm. ;  a  man  weighing  70  kgm.,  with  the 
same  susceptibility,  would  be  killed  by  0.23  mgm. 
Thirty  to  one  hundred  milligrams  of  strychnine  are 
required  to  kill  a  man. 


138  ATLAS   OF   BACTERIOLOGY. 


4.  Sulphuretted  Hydrogen, 

Sulphuretted  liydrogen  is  a  very  widely  distributed 
bacterial  product.  It  is  easily  demonstrated  by 
fastening,  by  means  of  the  cotton  plug,  a  moist  strip 
of  lead  acetate  paper  in  the  neck  of  the  culture  tube 
and  closing  it  with  a  rubber  cap  (made  of  black  rubber 
free  from  sulphur).  Frequent  observations  of  the 
originally  brownish,  later  black,  often  very  feeble  dis- 
coloration of  the  paper  is  necessary,  because  some- 
times the  color  fades  away  at  a  later  period.  Tests 
which  are  apparently  negative  should  not  be  ter- 
minated too  soon.  The  literature  consists  mainly  of 
articles  by  Petri  and  Maassen  (A.  G.  A.,  YIII.,  318 
and  490),  Eubner,  Stagnitta-Balistreri,  and  Niemann 
(A.  H.,  XVI.). 

Sulphuretted  hydrogen  may  be  formed  from : 

1.  Albuminoid  bodies.  (It  is  well  known  that 
mere  boiling  eliminates  H^S  from  egg  albumin). 
According  to  Petri  and  Maassen  this  power  inheres 
in  all  the  bacteria  examined  upon  a  fluid  nutrient 
medium  which  is  rich  in  peptone  (five  to  ten  per 
cent)  and  free  from  sugar;  in  bouillon  free  from 
peptone  very  few  varieties  form  H^S  (for  example, 
bacterium  vulgare);  in  bouillon  containing  one  per 
cent  peptone,  about  fifty  per  cent  of  the  bacteria 
(Stagnitta-Balistreri) . 

2.  Powdered  sulphur.  In  nutrient  media  to  which 
pure  powdered  sulphur  has  been  added  all  bacteria 
produce  much  larger  amounts  of  sulphuretted  hy- 
drogen than  without  this  addition.  Petri  and  Maas- 
sen regard  this  production  of  sulphuretted  hydrogen 


SUGAR   IN-   THE   NUTRIENT  MEDIUM.  139 

as  a  function  of  the  nascent  liydrogen  wliich  the  bac- 
teria produce,  i.e.,  they  regard  the  formation  of  H^S 
as  a  proof  of  the  formation  of  nascent  hydrogen. 

3.  Thiosulphate  and  thiosulphite.  This  has  been 
studied  especially  in  yeast  but  has  also  been  demon- 
strated in  the  case  of  some  bacteria  (by  Petri  and 
Maassen). 

4.  Sulphates.  Beyerinck  in  particular  has  de- 
monstrated this  practically  important  function  for  his 
(morphologically  poorly  characterized)  motile,  strict 
anaerobic  spirillum  desulphuricans.  It  is  rarely 
found  developed  among  other  bacteria  (C.  B.,  Part 
11. ,  Vol.  L,  1). 

Rubner  has  shown  that  in  bacterium  vulgare  the 
decomposed  organic  sulphur  always  suffices  for  the 
production  of  sulphuretted  hydrogen. 

The  presence  of  sugar  in  the  nutrient  media  rarely 
prevents  or  diminishes  the  production  of  sulphur- 
etted hydrogen,  even  when  the  bacteria  are  able  to 
decompose  (ferment)  sugar  vigorously.  The  decom- 
position of  hydrocarbons  does  not  protect  the  albumi- 
noids from  decomposition.  The  presence  of  saltpetre 
is  a  disturbing  factor  and  under  these  circumstances 
very  little  H^S  but  an  abundance  of  nitrite  is  formed 
(Petri  and  Maassen).  The  exclusion  of  oxygen  favors 
the  production  of  sulphuretted  hydrogen.  On  pass- 
ing air  through  the  cultures  of  facultative  anaerobic 
producers  of  sulphuretted  hydrogen  the  amount  of 
H^S  produced  diminishes  considerably,  and  in  its 
place  sulphates  are  formed. 

Many  producers  of  sulphuretted  hydrogen  also 
produce  stinking  mercaptan  (CH^— SH),  demonstra- 
ble by  tiie  green  color  which  it  gives  to  the  yellow- 


140  ATLAS   OF    BACTERIOLOGY. 

ish-red  isatin  sulphate.  Upon  the  culture  glass  is 
placed  a  tube  open  on  both  sides ;  this  is  filled  with 
glass  beads  which  are  moistened  with  a  one  and  a 
half  per  cent  solution  of  isatin  in  concentrated  sul- 
phuric acid.  The  presence  of  sugar  iu  the  nutrient 
media  diminishes  or  prevents  the  formation  of  mer- 
captan. 

5.  Reduction  Processes. 

(Keduction  of  Coloring  Matters,  Nitrates,  etc.) 

We  have  seen  that  aerobic  bacteria  in  general 
possess  the  power  of  converting  powdered  sulphur 
into  sulphuretted  hydrogen  and  that  nascent  hydrogen 
is  necessary  thereto. 

Similar  processes,  and  probably  also  due  in  part 
to  nascent  hydrogen,  are  the  following : 

1.  Reduction  of  blue  litmus  coloring  matter,  methyl 
blue,  and  indigo  when  added  to  colorless  leuco-prod- 
ucts.  The  upper  layer  in  contact  with  the  air  often 
shows  no  reduction,  only  the  deeper  layers.  On 
shaking  in  the  air  the  color  is  restored,  but  occasion- 
ally the  litmus  coloring  matter  is  restored  with  a  red 
color  on  account  of  the  coincident  production  of  acid. 
The  mode  of  experiment  goes  without  saying ;  bouil- 
lon serves  as  the  nutrient  medium.  According  to 
Cahen  the  reduction  of  litmus  is  effected  by  all  lique- 
fying bacteria.  It  is  observed  very  beautifully,  for 
example,  in  bacillus  fluorescens  liquefaciens,  but 
there  are  also  non-liquefying  varieties  (for  example, 
bacterium  coli)  which  exhibit  this  characteristic. 

2.  Reduction  of  nitrates  to  nitrites  and  ammonia. 
The  former  power  seems  to  belong  to  very  many  bac- 


SUGAR   IN^   THE   NUTRIENT   MEDIUM.  141 

teria.  At  least  Petri  and  Maassen  found  that,  among 
six  varieties  cultivated  in  bouillon  containing  2.5-5 
per  cent  peptone  and  0.5  per  cent  saltpetre,  there  was 
almost  always  a  pronounced  production  of  nitrites; 
in  one  case,  indeed,  only  ammonia  was  found.  Rub- 
ner  (A.  H.,  XYI.,  62)  found  the  production  of  nitrites 
absent  only  in  isolated  cases.  Among  twenty-five 
varieties  Warington  found  that  eighteen  produced 
nitrites.  In  our  experiments  with  bacterium  coli, 
typhi,  vulgare,  bacillus  anthracis,  subtilis,  vibrio 
cholerae,  the  addition  of  sugar  was  not  a  disturbing 
factor.  At  the  end  of  three  days  the  nitrite  reaction 
was  equally  pronounced,  with  or  without  the  pres- 
ence of  one  per  cent  grape  sugar,  in  one  per  cent  pep- 
tone bouillon  containing  one-half  per  cent  saltpetre. 

Nitrites  are  demonstrated  in  the  following  way: 
After  the  tubes  have  remained  for  a  few  days  in  the 
incubating  chamber,  some  potassium  iodide  starch 
solution  (thin  starch  paste  with  one-half  per  cent 
KI)  and  a  few  drops  of  sulphuric  acid  are  added  to 
the  nitrate  bouillon  (also  to  two  uninoculated  control 
tests).  The  control  tubes  remain  colorless  or  at  the 
most  gradually  acquire  a  very  faint  blue  color,  but 
if  nitrites  are  present,  a  dark  blue  to  dark  brownish- 
red  color  develops.  Small  amounts  of  nitrite  are  de- 
monstrated by  metaphenylendiamin  and  somewhat 
diluted  sulphuric  acid  (yellowish-brown  color)  or 
(most  clearly)  by  a  mixture  of  sulphanilic  acid  and 
naphthylamin  (red  color) .  ( Vide  Dieudonne,  A.  G. 
A.,  XL,  508). 

The  demonstration  of  ammonia  by  the  addition  of 
Nessler's  reagent  is  permitted  only  upon  inorganic 
non-saccharine  nutrient  media.      In  bouillon  Ness- 


142  ATLAS   OF   BACTERIOLOGY. 

ler's  reagent  is  reduced  almost  immediately  to  black 
mercurial  oxide.  A  strip  of  paper  which  has  been 
dipped  in  the  reagent  may  be  hung  over  bouillon 
cultures,  or  the  latter  may  be  distilled  after  addition 
of  MgO  and  the  distillate  treated  with  Nessler's  re- 
agent. A  yellow  to  reddish-brown  color  indicates  the 
presence  of  ammonia.     Control  tests  must  be  made. 

6.  Aromatic  Metabolic  Products. 

It  is  evident  that  albumin  gives  rise,  under  the  in- 
fluence of  very  many  varieties  of  bacteria,  to  aromatic 
bodies  of  which  indol,  skatol,  phenol,  and  tyrosin 
are  the  best  known.  Methodical  investigations  have 
been  made  only  in  regard  to  indol  and  phenol,  as 
these  bodies  are  easily  recognized. 

Demonstration  of  indol :  To  the  bouillon  culture — 
whicli  should  not  be  less  than  a  week  old  and  made 
without  any  addition  of  sugar — about  half  its  volume 
of  ten-per-cent  sulphuric  acid  is  added.  If  a  rose  to 
bluish-red  color  appears  forthwith  on  warming  to 
about  80°,  then  indol  and  nitrite  are  both  present,  as 
this  nitrosoindol  reaction  requires  the  presence  of 
both  bodies.  The  test  is  generally  successful  in 
cholera  and  other  vibriones  and  occasionally  in  diph- 
theria (red  cholera  reaction).  But  as  a  general  thing 
the  addition  of  sulphuric  acid  does  not  suffice,  and  it 
is  necessary  to  add  a  little  nitrite.  This  may  be  done 
later,  after  the  culture  has  been  heated  without 
nitrite,  and  no  reaction  or  a  very  doubtful  one  has 
been  obtained.  Of  the  solution  containing  about 
0.05  per  cent  sodium  nitrite  we  add  1  to  2  c.c.  until  the 
maximum  of  the  reaction  is  secured.  The  addition 
of    strong  nitrite    solutions  gives   the   acid  fluid   a 


SUGAR   IN^   THE   NUTRIEN"T  MEDIUM.  143 

brownish-yellow  color  and  prevents  entirely  the  de- 
monstration of  indol. 

Demonstration  of  phenol:  The  culture,  which  is 
made  in  non-saccharine  bouillon,  receives  about  one- 
fifth  its  volume  of  hydrochloric  acid  and  is  then  dis- 
tilled. The  distillate  deposits  flocculi  with  bromine 
water,  or  assumes  a  violet  color  on  the  addition  of 
calcium  carbonate  and  cautiously  neutralizing,  or  of 
neutral  very  dilute  ferric  chloride. 

Among  sixty  varieties  examined  we  found  indol 
formed  twenty-three  times,  and  our  findings  agree 
with  those  of  Levandovsky  (Deiitsch.  med.  Wschr., 
1890,  No.  51).  The  chief  indol  producers  are  the 
coli  group  in  the  widest  sense — glanders,  diphtheria, 
proteus,  and  the  majority  of  vibriones.  According 
to  Levandovsky  the  indol  producers  just  mentioned, 
with  the  exception  of  the  vibriones,  also  form  phenol. 
We  have  tested  the  production  of  phenol  only  in  bac- 
terium coli  and  vulgare  and  found  mere  traces  in  five- 
day  cultures. 

7.  Decomposition  of  Fats. 

Pure  melted  butter  is  not  a  nutrient  medium  for  bac- 
teria. The  rancidity  of  butter  is  due  to :  (1)  a  purely 
chemical  decomposition  of  the  butter  by  the  oxygen 
of  the  air,  aided  by  sunlight  (Duclaux,  Ritsert) ;  (2)  a 
lactic-acid  fermentation  of  the  milk  sugar  which  has 
been  left  over  in  the  butter  (vide  v.  Klecki,  C.  B., 
XV. ,  354) .  Finally  fat  is  attacked  by  bacteria  and  acid 
is  formed,  if  it  is  mixed  with  gelatin  as  a  nutrient 
medium  (pide  v.  Sommaruga,  Z.  H.,  XVII.,  441). 


144  ATLAS   OF   BACTERIOLOGY. 


8.  Putrefaction  (Appendix  to  1-7). 

Putrefaction,  in  the  language  of  the  laity,  means 
every  decomposition  which  is  produced  by  bacteria 
and  is  attended  by  the  formation  of  foul-smelling 
substances. 

On  scientific  investigation  it  is  found  that  the  al- 
buminoids and  their  allies  are  the  substratum  of  pu- 
trefaction; at  first  they  are  often  peptonized,  then 
they  are  split  up  still  further. 

Typical  putrefaction  occurs  only  when  the  supply 
of  oxygen  is  wanting  or  scanty.  The  vigorous  pas- 
sage of  air  through  a  putrefaction  bacteria  culture— a 
process  which  does  not  occur  in  natural  putrefaction 
— modifies  the  process  in  the  most  marked  manner. 
In  the  first  place  because  the  anaerobic  putrefac- 
tion bacteria  are  killed  or  their  growth  is  inhibited, 
and  secondly  by  the  action  of  the  oxygen  upon  the 
products  or  intermediate  products  of  the  aerobic  and 
facultative  anaerobic  bacteria.  Finally,  it  seems 
conceivable  that  the  same  bacteria  (anaerobic  and 
aerobic)  may  from  the  start  furnish  different  products 
of  putrefaction. 

Among  the  putrefaction  products  we  find  the  bod- 
ies *  described  in  preceding  chapters :  peptone,  am- 
monia and  amins,  leucin,  tyrosin  and  other  amido 
bodies,  oxyfatty  acids,  indol,   skatol,   phenol,  finally 

*It  is  often  said  that  in  every  putrefaction  the  albuminoid 
bodies  are  first  peptonized,  but  inasmuch  as  bacterium  vulgare  /3 
Zcnkeri,  and  bacterium  putidum  are  generally  recognized  as  pro- 
ducers of  putrefaction,  and  as  they  do  not  even  liquefy  gela- 
tin, we  cannot  always  speak  of  peptonization  of  albumin  as 
constant  in  putrefaction. 


SUGAR   IN  THE   NUTRIENT   MEDIUM.  145 

sulphuretted  hydrogen,    mercaptan,   carbonic    acid, 
hydrogen,  marsh  gas. 

But  inasmuch  as,  in  putrefaction  of  different  nu- 
trient media  by  different  bacteria,  the  metabolic 
products  just  mentioned  are  found,  as  a  rule,  only  in 
part  and  in  extremely  varying  combinations,  putre- 
faction can  hardly  be  defined  more  accurately  with 
chemical  aids  than  is  possible  with  the  senses. 
Hence  I  believe  it  is  best  to  employ  the  term  putre- 
faction only  in  the  general  lay  signification  of  every 
foul-smelling  decomposition  of  albuminoids  (vide 
Kuhn:  A.  H.,  XIII.,  1). 

9.  Nitrification. 

The  formation  of  small  amounts  of  nitrous  and 
nitric  acids  is  widely  diffused  among  bacteria. 
Heraeus  (Z.  H.,  1,  193),  who  first  investigated  the 
subject  with  pure  cultures,  found  that  in  sterilized 
urine  which  had  been  diluted  fourfold  very  many  of 
the  well-known  bacteria  form  small  amounts  of  nitrite 
from  urea  or  ammonium  carbonate.  These  include 
micrococcus  pyogenes  citreus,  bacterium  prodigi- 
osum,  typhi,  coli,  bacillus  mycoides,  anthracis, 
vibrio  pyogenes,  and  vibrio  proteus.  Various  soil 
bacteria  also  furnish  nitrites.  The  addition  of  sugar 
interferes  with  the  production  of  nitrite  from  NHg 
until  it  is  destroyed.  The  formation  of  nitrate  was 
not  studied  by  Heraeus.  Warington  failed  to  find 
nitrates  in  a  study  of  twenty-four  varieties  in  pure 
cultures  in  nutrient  solutions  which  formed  nitrate 
distinctly  when  infected  by  means  of  the  soil  (C.  B., 
YI.,  498). 

According  to  more  recent  investigations  nitrifica- 
10 


146  ATLAS   OF   BACTERIOLOGY. 

tion  is  particularly  the  function  of  a  small,  special 
group  of  bacteria  which  are  cultivated  with  difficulty 
and  do  not  thrive  upon  our  ordinary  nutrient  media. 

According  to  Winogradsky,  who  has  done  the  most 
work  in  this  department,  the  facts  of  the  case  are  as 
follows :  The  soil  of  Europe  contains,  widely  distrib- 
uted, two  micro-organisms,  one  of  which  (nitroso- 
monas)  converts  ammonia  into  nitrite,  the  other  (called 
nitromonas,  later  nitrobacter)  converts  nitrite  into 
nitrate.  Both  varieties  are  obtained  mixed  when  bits 
of  earth  in  flasks  are  dissolved  in  boiling  water 
(Winogradsky  took  the  water  of  a  fresh-water  lake) 
containing  1  gm.  ammonium  sulphate  and  1  gm.  potas- 
sium phosphate  to  1  litre.  About  1.0  gm.  basic  mag- 
nesium carbonate  is  added  to  each  flask  containing 
100  c.c.  Considerable  development  of  nitrites  takes 
place,  and  gradually  nitrates  are  also  formed.  By 
inoculation  of  new  flasks  the  nitrifying  organisms 
are  obtained  gradually  in  a  purer  state,  and  silicic- 
acid  plates  finally  i)ermit,  with  difficulty,  a  pure  cul- 
ture. Burri  and  Stutzer  have  recently  cultivated  upon 
the  ordinary  nutrient  media  a  vigorous  nitrate  pro- 
ducer (from  nitrite),  but  it  forms  nitrates  only  upon 
inorganic  nutrient  solutions  (C.  B.,  Vol.  I.,  Part  II., 
731). 

P.  F.  Kichter  (C.  B.,  XYIII.,  Part  I.,  p.  129)  ob- 
served on  several  occasions  a  pronounced  nitrite 
reaction  in  fresh  urine  evacuated  with  the  catheter. 
From  one  specimen  he  isolated  a  coccus  of  medium 
size,  which  in  twenty  minutes  produced  a  very  in- 
tense nitrite  reaction  in  fresh  urine.  In  addition  it 
reduced  nitrate  to  nitrite. 


SUGAR   IN  THE   ITUTRIENT  MEDIUM.  147 


10.   Conversion  of  Nitrons  and  Nitric  Acids  into  Free 
Nitrogen. 

This  process  is  carried  on  by  an  entire  series  of 
bacteria.  Burri  and  Stutzer  (C.  B.,  Part  II.,  Yol. 
I.,  No.  7  et  seq.)  were  the  first  to  describe  special  ni- 
trate fermenters  in  such  an  accurate  manner  that  they 
could  again  be  recognized.  They  first  isolated  from 
horse  manure  two  bacteria,  of  which  each  alone  was 
unable  to  produce  nitrogen  from  nitrate,  but  did  this 
vigorously  when  combined,  and  when  the  supply  of 
oxygen  was  abundant  or  scanty  but  never  when  it  was 
absent.  These  two  synergetic  bacteria  are :  (1)  Bac- 
terium coli  (this  may  be  replaced  by  bacterium  typhi) , 
and  (2)  a  short  rod  described  as  bacillus  denitri- 
ficans  I.  Later  these  writers  found  a  bacillus  deni- 
trificans  II.,  which  alone  effected  the  entire  decom- 
position of  nitrate  into  nitrogen.  We  found  that 
bacterium  pyocyaneum  also  converts  saltpetre  into 
nitrogen. 

The  practical  importance  of  these  organisms  lies 
in  the  fact  that  through  their  agency  considerable 
amounts  of  nitrates  in  the  soil,  but  particularly  in 
manures,  may  be  lost  for  the  nourishment  of  plants 
on  account  of  their  conversion  into  nitrogen. 

11.  Assimilation  of  Nitrogen. 

According  to  our  present  knowledge  no  other  vege- 
table family  is  able  to  assimilate  the  nitrogen  of  the 
air,  but  this  power  does  inhere  in  one  form  of  bac- 
teria, the  bacillus  radicicola  Beyerinck.  This  bac- 
terium is  found  in  the  small  root  knobs  of  various 


148  ATLAS   OF   BACTERIOLOGY. 

leguminosse  and  may  be  cultivated  from  them.  From 
the  different  forms  of  leguminosse  we  obtain  different 
races  of  the  bacteria,  each  one  being  especially 
adapted  to  one  form  of  leguminosse;  not  every  race 
is  able  to  produce  the  knobs  in  every  form  of  the 
vegetable.  There  are  also  "neutral"  bacteria,  found 
free  in  the  soil,  which  are  not  specially  adapted  to 
any  form  of  the  leguminosse  and  which  are  able  to 
produce  knobs  in  very  different  forms  of  the  vege- 
table. 

With  the  aid  of  these  root  knobs,  which  are  due  to 
the  immigration  of  the  root  bacteria,  the  leguminosse 
are  able  to  furnish  crops  which  are  rich  in  nitrogen 
from  a  relatively  sterile  soil  which  is  very  poor  in  ni- 
trogen. The  manner  in  which  the  absorption  of 
nitrogen  takes  place  is  still  entirely  unknown.  It  is 
claimed  that  the  swollen  zoogloea  form  of  bacteria 
(bacteroids*),  almost  always  observed  in  the  knobs, 
is  alone  able  to  absorb  nitrogen.  Recently  it  seems  to 
have  been  j^roven  that  even  without  the  aid  of  legu- 
minosse  knob  bacteria  living  free  in  the  soil  are  able 
to  absorb  elementary  nitrogen  (for  a  detailed  rhume 
of  the  present  status  of  the  question,  see  Stutzer :  C. 
B.,  PartlL,  Yol.  I.,  p.  8). 

12,  Production  of  Acids  from  Carbohydrates. 

As  Theobald  Smith  showed  (C.  B.,  XVIII.,  No.  1), 
the  formation  of  free  acid  is  only  possible  on  a 
saccharine  nutrient  medium.  Its  production  upon 
ordinary  bouillon  takes  place  only  when  the  latter 

*  These  bacteroids  assume  the  most  bizarre  shapes,  networks, 
forks,  stars. 


SUGAR   IN^   THE   NUTRIENT  MEDIUM.  149 

contains  grape  sugar  (derived  from  the  meat).*  Ac- 
cording to  Smith  all  strict  or  facultative  anaerobics 
form  acids  out  of  sugar,  the  aerobics  either  do  not 
or  they  do  it  so  slowly  that  the  formation  of  the  acid 
is  concealed  by  the  parallel  production  of  alkali. 
Prior  to  a  knowledge  of  this  work  we  had  found  that 
all  tested  varieties  of  bacteria  (about  sixty),  which 
are  shown  in  the  Atlas,  formed  more  or  less  free 
fixed  acid  in  one  per  cent  grape  sugar  peptone 
bouillon  (vide  Table  I).  The  formation  of  acid 
may  or  may  not  be  attended  with  visible  develop- 
ment of  gas.  Intense  production  of  acid  may  kill 
the  cultures  (for  example,  bacterium  coli,  vulgare, 
etc.). 

In  many  varieties  the  formation  of  acid  or  decom- 
position of  sugar  is  intense  and  rapid,  so  that  this 
metabolism,  which  is  effected  chiefly  at  the  expense 
of  the  carbohydrates,  is  called  fermentation.  Inas- 
much as  this  is  attended  not  infrequently  by  the  de- 
velopment of  gas  in  large  quantity,  this  term  also 
seems  justifiable  to  the  laity. 

If,  after  the  sugar  is  used  up,  the  amount  of  acid 
produced  is  insufficient  to  kill  the  bacteria,  the  metab- 
olism which  ensues  is  that  common  to  the  non-sac- 
charine nutrient  medium,  the  acid  is  gradually  neu- 
tralized, and  finally  an  increasing  alkaline  reaction 
sets  in. 

Among  the  acids  produced  (apart  from  carbonic 
acid,  which  will  be  considered  under  the  heading  of 
"Production  of  Gas")  the  most  important  and  widely 
distributed   is  lactic   acid.     In   addition  we  almost 

*  According  to  Th.  Smith,  seventy -five  per  cent  of  commercial 
beef  contains  distinct  amounts  of  sugar  (up  to  0.3  per  cent). 


150  ATLAS   OF   BACTERIOLOGY. 

always  find,  at  least  in  traces,  formic  acid,  acetic 
acid,  proprionic  acid,  butyric  acid,  and  not  infre- 
quently some  ethyl  alcohol,  aldehyde,  or  acetone.  In 
rarer  cases  the  lactic  acid  is  wanting  and  only  the 
other  acids  are  formed. 

In  order  to  obtain  and  separate  the  acids  we 
employ  the  following  method:  In  1  litre  flasks  are 
placed  i  litre  peptone  bouillon  with  two  to  ^yq  per 
cent  grape  sugar  or  milk  sugar  and  perhaps 
10  gm.  calcium  carbonate.  The  acids  formed  com- 
bine with  the  calcium  carbonate  into  a  soluble 
lime  salt  and  carbonic  acid  escapes;  the  reaction 
of  the  fluid — and  that  is  the  main  thing — remains 
neutral.  A  strongly  acid  reaction  would  inter- 
fere prematurely  with  the  further  growth  of  the 
bacteria. 

When  the  growth  has  ceased  (in  eight  to  fourteen 
days)  the  undissolved  carbonate  is  filtered  off,  and 
the  reaction  being  neutral,  the  alcohol,  aldehyde, 
acetone,  etc.,  are  distilled;  the  fluid  is  boiled  down 
considerably  during  this  process.  The  three  sub- 
stances just  mentioned  are  detected  in  common  by 
Lieben's  iodoform  reaction.  To  the  slightly  warmed 
fluid  in  a  test  tube  are  added  five  to  six  drops  of 
pure  ten-per-cent  potash  lye,  then  a  weak  iodine- 
potassium  iodide  solution  is  added  drop  by  drop 
until  a  brown  color  is  produced,  and  the  latter  is 
made  to  disappear  by  a  drop  of  potash.  The  charac- 
teristic iodoform  odor  and  the  precipitation  of  micro- 
scopic small  six-angled  iodoform  plates  are  convinc- 
ing evidence.  For  the  differentiation  of  alcohol, 
aldehyde,  and  acetone,  vide  Yortmann,  "  Analyse  or- 
gan. Stoffe,"  1891. 


SUGAR  IK  THE   NUTRIENT  MEDIUM.  151 

A  strong  acid  reaction  is  now  secured  with  phos- 
phoric acid  and  the  volatile  acids  are  distilled  off 
with  the  aid  of  a  current  of  steam.  The  distillation 
must  be  prolonged  because  the  complete  removal  of 
the  volatile  acids  is  difficult.  The  lactic  acid  is  left 
in  the  distillate,  is  obtained  by  shaking  repeatedly 
with  pure  ether,  and  the  ether  is  then  distilled  off. 

The  lactic  acid  obtained  is  always  ethylidenlactic 
CH3 

acid  CHOH,  which  occurs  in  two  stereoisomeric  forms : 

COOH 

(1),  dextro-rotatory  with  laevo-rotatory  zinc  salt,  and 
(2)  Isevo-rotatory  with  dextro-rotatory  zinc  salt.  If, 
as  happens  very  frequently,  exactly  the  same  number 
of  molecules  of  left  and  right  lactic  acids  are  present, 
then  the  combination  is  optically  inactive  and  forms 
the  so-called  "fermentation  lactic  acid."  1  assume 
that  both  lactic  acids  often  develop  from  sugar,  but 
that  some  varieties  of  bacteria  thrive  mainly  on  one, 
some  on  the  other  form,  so  that  sometimes  there  is  a 
uniform  combination,  sometimes  one  form  predom- 
inates or  alone  remains. 

Since  Schardinger  {Mitt.  f.  Chem.,  XI.,  545)  dis- 
covered that  the  previously  unknown  left  lactic  acid 
was  the  product  of  a  short  rod  bacillus  in  water,  the 
pupils  of  Nencki  and  Kubner  have  made  numerous 
investigations  on  the  lactic  acids  formed  by  the  dif- 
ferent varieties,  in  the  hope  of  utilizing  the  results 
for  purposes  of  differential  diagnosis. 

For  the  method  of  determining  the  form  of  lactic 
acid,  vide  Nencki  (C.  B.,  IX.,  305)  and  Gosio  (A.  H., 
XXL,  115). 


152  ATLAS   OF   BACTERIOLOGY. 

The  most  important  results  of  the  investigations 


are: 


Bacterium  coli 

Bacterium  Bischleri 

Bacterium  typhi  

Micrococcus  acidi  paralactici  . 

Vibrio  cholerse  (Calcutta) 

Vibrio  cliolerse  (Massaua)  . . . . 

Vibrio  Metschnikovi 

Vibrio  danubicus 

Vibrio  "Wernicl^e"  L,II.,I.-I. 

Vibrio  "Dunbar" 

Vibrio  proteus 

Vibrio  Weibel 

Vibrio  Bonhoff  b 

Vibrio  berolinensis 

Vibrio  aquatilis 

Vibrio  tyrogenes 

Vibrio  Bonhofl:  a 


Inactive 
lactic  acid. 


+ 


+ 


Right  lactic 

acid  = 

paralactic 

acid. 


+ 

+ 


+ 


Left  lactic 
acid. 


+ 


+ 


Although  these  results  are  not  yet  of  much  impor- 
tance, a  continuance  of  these  theoretically  interesting 
studies  is  desirable. 

Various  bacteria— which  have  in  great  part  been 
imperfectly  studied  morphologically  and  biologically 
— are  able  to  produce  butyric  acid,  butyl  alcohol,  or 
both  from  carbohydrates. 

A  review  of  these  varieties  is  found  in  an  article  by 
Baier  (C.  f.  B.,  Part  II.,  Yol.  I.,  p.  17).  Here  we 
will  only  mention :  bacillus  butyricus  Hiippe  (appar- 
ently also  other  aUied  varieties),  the  imperfectly 
described  granulobacter  poly  my  xa  Beyerinck,  and 
several  anaerobic  varieties  (Clostridium  butyricum  of 
the  authors). 

In  connection  with  the  fermentation  of  sugar  we 


SUGAR   IN  THE  NUTRIENT  MEDIUM.  153 

may  mention  tlie  splitting  up  of  cellulose  by  various 
bacteria,  which  are  found  particularly  in  the  gastric 
and  intestinal  contents  of  herbivora,  and  in  muck, 
and  which  form  marsh  gas  as  a  striking  product. 

Unfortunately  the  decomposition  of  cellulose  by 
bacteria  has  been  imperfectly  studied.  It  appears  to 
be  certain,  however,  that  at  least  one  anaerobic  variety' 
decomposes  cellulose  into  marsh  gas  and  carbonic 
acid.  But  the  most  recent  investigator  of  this  ques- 
tion. Van  Senus,  maintains  that  the  anaerobic  bacil- 
lus amylobacter  isolated  by  him  will  attack  cellulose 
only  in  symbiosis  with  another  small  bacillus  (vide 
the  resumS  by  Herzfeld:  C.  B.,  Part  I.,  Vol.  II.,  p. 
114). 

13.  Formation  of  Gas  from    Carbohydrates  and  other 
Fermentihle  Fatty  Bodies. 

The  only  gas  which  develops  in  visible  amounts 
upon  a  non-saccharine  nutrient  medium  is  nitrogen. 
If  sugar  is  vigorously  attacked  by  bacteria,  the  devel- 
opment of  gas  may  be  lacking  inasmuch  as  pure  lactic 
or  acetic  acid  is  produced  (for  example,  typhus 
bacillus  on  grape  sugar) ;  but  very  often  there  is  a 
notable  development  of  gas,  especially  when  the  air 
is  excluded.  About  one-third  of  the  varieties  which 
form  acid  vigorously  also  produce  an  abundance  of 
gas.  This  consists  of  carbonic  acid  which,  accord- 
ing to  Smith  (C.  B.,  XYIII.,  1)  is  always  combined 
with  hydrogen.  Marsh  gas  appears  to  be  formed 
rarely  (apart  from  the  bacteria  which  decompose  cel- 
lulose). Last  year  Mr.  Conrad  isolated  in  my  labor- 
atory, a  bacterium  allied  to  bacterium  coli,  which 
gives   rise   to   the   fermentation  of    sauerkraut  and 


154 


ATLAS   OF   BACTERIOLOGY. 


always,  even  when  the  nutrient  medium  is  free  from 
cellulose,  forms  some  marsh  gas  in  addition  to  car- 
bonic acid  and  hydrogen. 

In  order  to  determine  whether  gas  is  formed,  we 
should  use  the  agitation  culture  on  one-per-cent  grape 


Fig.  11.— Bacterium  Coli  upon  Sugar  Agar  at  the  end  oflVelve,  Twenty- 
four,  and  Forty -eight  Hours. 

sugar  agar.  At  the  end  of  twenty -four  hours  (often 
at  the  end  of  eight  to  twelve  hours  when  incubating 
temperature  may  be  employed)  the  agar  is  infiltrated 
with  bubbles  of  gas  or  even  split  up  by  numerous 
deep  rifts  and  fissures.  If  the  gas  is  to  bo  measured 
or  analyzed,  it  is  best,  according  to  Th.  Smith,  to  re- 
ceive it  in  the  fermentation  flask  which  has  been  used 


SUGAR   IN"  THE   NUTRIENT   MEDIUM. 


155 


^\ 


for  such  a  long  time  in  physiological  and  pathologi- 
cal chemistry. 

The  tubes,  which  should  have  the  shajje  shown  in 
Fig.  12,  are  filled  with  one-per-cent  grape  sugar  pep- 
tone bouillon  and  sterilized  in  the 
steam  chamber.  After  inoculation 
with  a  platinum  loop  in  the  incubat- 
ing chamber  the  following  facts  de- 
velop : 

1.  If  the  opacity  is  produced  only 
in  the  open  spherical  part  of  the  flask, 
we  have  to  deal  with  an  aerobic  va- 
riety; if  produced  only  in  the  closed 
tube  and  the  globe  remains  clear,  we 
have  to  deal  with  an  anaerobic  variety. 

2.  The  daily  amount  of  gas  pro- 
duced is  marked  with  ink ;  if  the  cali- 
bre of  the  tube  is  known,  we  are  able 

to  state,  after  the  formation  of  gas  has  ceased  on 
the  fourth  to  sixth  day,  what  percentage  of  gas  was 
produced  on  each  day. 

3.  A  rough  analysis  of  the  gas  should  be  made. 
After  the  amount  of  gas  has  been  noted,  we  fill  the 
open  sphere  completely  with  ten-per-cent  soda  lye, 
close  it  firmly  with  the  thumb,  and  shake  it  for  a 
while.  At  the  end  of  two  minutes  all  the  gas  is  al- 
lowed to  pass  into  the  closed  tube,  and  after  the 
thumb  is  removed  the  new  volume  of  gas  is  read  off. 
The  part  which  has  disappeared  is  carbonic  acid,  the 
rest  is  nitrogen,  hydrogen,  and  marsh  gas.  The  quan*- 
titative  analysis  of  these  gases  is  best  done  by  means 
of  Hempel's  gas  pipettes  [vide  Winkler:  "Lehrb. 
d.  techn.  Gasanalyse,"  Freiburg,  1892).     The  method 


Fig.  12.— Fermen- 
tation Flask. 


156  ATLAS   OF   BACTERIOLOGY. 

is  based  on  the  fact  that  hydrogen,  when  mixed  with 
oxygen  and  passed  over  glowing  palladium  asbestos, 
is  converted  into. water  and  accordingly  disappears; 
carburetted  hydrogen  is  changed  into  carbonic  acid 
in  a  glowing  platinum  capillary  tube,  and  is  measured 
as  such,  and  the  remainder  is  nitrogen.  With  some 
practice  the  examination  is  easy  and  accurate. 

14.    Production  of   Acids  from    Alcohols    and   other 
Organic  Acids. 

It  has  long  been  known  that  bacterium  aceti  or  its 
nearest  allies  convert  weak  solutions  of  ethyl  alcohol 
into  acetic  acid,  at  the  same  time  using  up  a  large 
amount  of  oxygen : 

JH^OH  COOH 


cii 


Higher  alcohols,  such  as  glycerin,  dulcite,  and 
mannite,  are  also  converted  into  acids;  glycerin  as 
generally  as  sugar  (v.  Sommaruga:  Z.  H.,  XY.,  291). 

Finally,  numerous  observations  have  been  made  on 
the  conversion,  b}^  bacteria,  of  acids  of  the  fatty 
series  (or  their  salts)  into  other  fatty  acids,  but  unfor- 
tunately the  majority  were  not  made  with  pure  cul- 
tures which  meet  the  modern  requirements.  Lactate, 
mallate,  citrate,  and  gly cerate  of  lime  were  usually 
employed  as  the  material  and  almost  always  acid 
mixtures  were  obtained  as  the  result  of  the  bacterial  ac- 
tivity. Among  these  butyric,  propionic,  valerianic, 
and  acetic  acids  play  the  principal  part;  succinic 
acid  and  ethyl  alcohol  are  often  found ;  formic  acid  is 
rarer.  Among  the  gases  carbonic  acid  and  hydrogen 
are  especially  prominent. 


SUGAR   IN   THE    NUTRIEI^^T   MEDIUM.  157 

Such  experiments  were  formerly  made  chiefly  by 
Fitz,  and  recently  have  been  performed  with  pure 
cultures  and  interesting  results  by  P.  Frankland.  A 
couple  of  illustrations  will  suffice.  Pasteur  found 
that  anaerobic  bacteria  convert  lactate  of  lime  into 
butyrate  of  lime. 

2(CH3  -  CHOH  -  C00)2Ca  =     COaCa  +  3  CO^  +  4  H^  + 
Lactate  of  lime.  (CH3-  CH2-CH2-  COO)Ca 

Butyrate  of  lime. 

According  to  P.  Frankland  the  bacillus  ethaceticus 
Fitz  converts  gly  cerate  of  lime  (CH^OH— CHOH— 
C00)2Ca  into  ethyl  alcohol,  acetic  acid,  carbonic 
acid,  and  hydrogen. 

in.  The  Pathogenic  Effects  of  Bacteria. 

(Pathogenesis,    Predisposition,  Resistance,  Im- 
munity.) 

Whenever  we  are  able  to  recognize  the  nature  of  the 
pathogenic  action  of  bacteria,  they  are  found  to  act 
by  means  of  the  chemical  substances  which  they  form 
in  the  animal  body  or  which  are  formed  from  them. 
But  hitherto  we  have  learned  to  comprehend  only  the 
action  of  those  bacteria  which  produce  toxic  sub- 
stances in  cultures,  and  by  means  of  which  we  can 
reproduce  the  characteristic  symptomatology  in  a 
more  or  less  accurate  manner. 

The  bacteria  of  this  category  include  particularly 
the  bacillus  tetani,  bacillus  diphtheriae,  streptococcus 
pyogenes,  micrococcus  pyogenes,  vibrio  cholerae,  etc. 
On  page  134  we  have  given  a  brief  sketch  of  our 
chemical  knowledge  of  these  toxic  substances. 


158  ATLAS   OF   BACTERIOLOGY. 

In  an  entire  series  of  important  infectious  dis- 
eases, on  the  other  hand,  we  are  almost  entirely  un- 
able to  explain  them  on  a  chemical  basis.  These 
include  anthrax,  rabbit  septicaemia,  hog  erysipelas. 
Filtrates  through  i)orcelain  of  the  most  virulent 
cultures  are  inert ;  the  cultures,  which  are  cautiously 
killed  by  briefly  warming  them  or  by  a  short  expo- 
sure to  chloroform,  produce  only  the  general  protein 
action  (fever)  when  injected.  Yet  it  is  probable  that 
even  these  diseases  are  toxaemias  due  to  bacterial 
metabolic  products. 

It  is  to  be  regarded  as  an  important  finding  that 
Petri  and  Maassen  (A.  G.  A.,  YIII.,  318)  were  able 
to  demonstrate  the  sulphmethsemoglobin  strii)e  in 
the  fresh  blood  and  oedema  fluid  of  erysipelatous  hogs 
— a  sign  that  poisoning  with  sulphuretted  hydrogen 
at  least  plays  a  part  in  the  death  of  the  animals. 
Similar  evidence  has  also  been  obtained  in  malignant 
oedema. 

Hoffa  regards  rabbit  septicaemia  as  methylguanidin 
poisoning  (Langenbeck's  Arch.,  1889,  p.  273).  Em- 
merich and  Tsuboi  {31unch.  med.  Wschr.,  1893,  No. 
25)  explain  cholera  as  a  nitrite  poisoning,  but  this 
has  been  vigorously  opposed. 

These  explanations  are  very  interesting,  but  they 
do  not  seem  to  suffice,  inasmuch  as,  apart  from  the 
toxic  processes  just  mentioned,  there  are  at  least 
other  speciflc  processes  in  the  blood  and  tissues  of 
the  animal.  This  is  proven,  among  other  things,  by 
the  development  of  specific  protective  substances 
("anti -bodies"). 

In  order  that  a  pathogenic  action  may  be  observed 
the  micro-organism  must  be  in  a  condition  of  vigorous 


SUGAR   IN  THE   NUTRIENT  MEDIUM.  159 

virulence,  the  inoculation  must  be  made  upon  a  sensi- 
tive animal,  and  the  proper  channel  of  infection  must 
be  selected. 

The  virulence  of  bacteria  varies  like  all  their  other 
functions  (production  of  coloring  matters,  fermenta- 
tion, etc.),  and  is  best  retained  by  constant  inocula- 
tion from  one  sensitive  animal  to  another.  This  is 
also  done  in  many  varieties  by  tolerably  frequent 
transmission  (about  once  a  month)  from  one  artificial 
nutrient  medium  to  another,  preferably  with  an  occa- 
sional intermediate  inoculation  of  an  animal.  On 
the  other  hand,  the  virulence  usually  suffers  when,  on 
account  of  rare  inoculations,  the  cultures  remain  for  a 
long  time  in  contact  with  their  accumulating  meta- 
bolic products. 

Attenuation  of  the  virulence  is  easily  effected : 

(a)  By  making  the  cultures  at  somewhat  too  high 
a  temperature.  For  example,  at  42.5°  anthrax  is  en- 
tirely deprived  of  virulence  in  three  to  four  weeks,  at 
47°  in  a  few  hours,  at  50°-53°  in  a  few  minutes.  By 
the  proper  regulation  of  the  action  of  heat  iihe  bacil- 
lus anthracis  may  be  attenuated  to  such  a  degree  that 
it  will  kill  only  mice,  or  mice  and  guinea-pigs,  or 
these  animals  and  rabbits. 

Spores  may  also  be  "attenuated"  by  dry  heat  or 
brief,  careful  disinfection  with  steam. 

{b)  By  cultures  upon  an  unsuitable  nutrient  me- 
dium. The  addition  of  phenol  (1 :  600),  potassium 
bichromate  (0.04-0.02  per  cent)  was  emi)loyed  suc- 
cessfully to  attenuate  bacillus  anthracis,  iodine  tri- 
chloride to  attenuate  the  bacilli  of  diphtheria. 

(c)  By  the  action  of  sunlight,  compressed  oxy- 
gen, etc. 


160  ATLAS   OF    BACTERIOLOGY. 

(d)  By  repeated  inoculation  of  unsuitable  animals. 
The  bacilli  of  hog  erysipelas  become  much  less  viru- 
lent from  passing  repeatedly  through  the  rabbit;  the 
organisms  of  variola  (these  are  not  bacteria)  from 
passing  through  the  body  of  the  cow. 

It  is  much  more  difficult  to  increase  the  virulence 
of  those  bacteria  which  have  been  attenuated.  On 
the  whole,  it  may  be  said  that  the  virulence  returns 
spontaneously  so  much  more  readily  the  more  rapidly' 
the  attenuation  has  been  effected. 

Varieties  which  have  slowly  and  spontaneously  lost 
their  virulence  may  often  be  restored  to  increased 
virulence  in  the  following  ways : 

1.  Culture  in  bouillon  to  which  ascites  fluid  has 
been  added  (streptococci,  diphtheria). 

2.  We  first  infect  especially  sensitive  animals — par- 
ticularly very  young  ones,  such  as  young  guinea-pigs 
—and,  when  these  have  succumbed,  convey  the  germs 
(directly  with  the  blood  of  the  animals)  to  older  and 
more  resistant  animals  of  the  most  sensitive  species, 
later  to  more  resistant  species.  Each  passage  through 
an  animal  increases  the  virulence  until  finally  a  certain 
maximum  is  reached. 

3.  Sensitive  animals  are  infected  with  large  amounts 
of  the  fresh  bouillon  culture  of  the  bacteria.  The 
metabolic  products,  which  are  introduced  at  the  same 
time,  then  increase  the  predisposition  for  the  injected 
organism. 

4.  Large  amounts  of  the  metabolic  products  of  bac- 
terium vulgare  are  injected  with  the  bacteria  (this  has 
been  especially  useful  in  the  case  of  staphylococci 
and  streptococci).  The  explanation  of  the  effect  is 
the  same  as  that  of  3. 


SUGAR   IN  THE   NUTRIENT  MEDIUM.  161 

5.  We  inject — for  example,  with  the  attenuated 
bacillus  oedematis  maligni  or  anthracis — another 
variety  which  per  se  is  almost  entirely  harmless — for 
example,  bacterium  prodigiosum. 

6.  We  inject  the  culture,  mixed  with  an  injurious 
substance  of  non-bacterial  origin — for  example,  lactic 
acid.  In  bacillus  oedematis  maligni  this  has  pro- 
duced increased  pathogenic  power,  probably  from 
local  impairment  of  the  anti-bacterial  activity  of  the 
animal  at  the  site  of  inoculation. 

The  susceptibility  of  different  species  of  animals 
and  of  different  individuals  to  different  infectious 
diseases  varies  from  birth  in  a  striking  and  not  easily 
explained  manner. 

Certain  species  are  absolutely  immune  against  spe- 
cial infection-producers.*  For  example,  man  against 
rinder  pest,  the  cow  against  glanders,  and  all  ani- 
mals which  have  been  tested  against  syphilis,  malaria, 
and  gonorrhoea. 

A  series  of  other  diseases  is  conveyed  very  rarely 
and  with  difficulty  to  certain  animals — for  example, 
anthrax  to  certain  varieties  of  pigeons,  rats,  and 
sheep.  This  constitutes  relative  immunity.  The 
more  vigorous  and,  as  a  general  thing,  the  more 
mature  an  animal  is,  the  more  completely  is  its  rela- 
tive immunity  developed.  Noxious  influences  of  all 
kinds  (hunger,  cold,  excessive  exertion,  ingestion  of 
*  It  is  especially  remarkable  that  very  closely  allied  varieties 
often  exhibit  astonishing  differences.  For  example,  the  glanders 
bacillus  can  be  conveyed  very  readily  to  the  field  mouse  but  not 
to  the  house  mouse  ;  the  bacillus  anthracis  kills  the  house  mouse 
with  almost  absolute  certainty  and  is  hardly  pathogenic  to  the  rat. 
Micrococcus  tetragenus  is  pathogenic  to  the  white  variety  of  the 
house  mouse,  but  is  not  virulent  to  the  gray  variety. 
11 


162  ATLAS    OF   BACTERIOLOGY. 

certain  poisons)  diminish  the  immunity  to  a  consider- 
able extent,  so  that  a  large  number  of  organisms 
which  are  weakened  in  this  manner  succumb  to  a 
subsequent  infection. 

Hence  in  every  newly  isolated  variety  of  bacteria 
whose  pathogenic  action  we  desire  to  prove  it  is 
necessary  to  experiment  ui)on  various  animals  if  the 
experiments  on  those  first  selected  proved  negative. 
The  principal  animals  for  experimentation  are :  the 
white  domestic  mouse,  white  rat,  guinea  pig,  rab- 
bit, chicken,  pigeon,  and,  for  special  purposes,  the 
monkey.  More  rarely  we  emj^loy  the  gray  domestic 
mouse  and  rat,  field  mice,  dogs,  cats,  cows,  sheep, 
pigs,  and  horses.  The  most  convenient  animal,  but 
one  requiring  good  care,  is  the  guinea-pig,  charac- 
terized by  suitable  size,  mildness,  and  modest  con- 
sumption of  food.  Animal  plagues  are  studied  and 
explained  much  more  readily  than  human  diseases, 
because  the  animals  are  at  our  disposal  for  experi- 
mentation. In  difiicult  cases  various  experiments  in 
infection  have  also  been  made  upon  man. 

The  causes  of  congenital  immunity  (resistance) 
reside  in  protective  arrangements  of  the  organism 
which  I  cannot  here  consider  in  detail.  It  may  be 
said,  however,  that  the  views  formulated  by  Buchner 
as  a  compromise  between  the  various  opposing 
theories  are  in  tolerable  accord  with  all  the  facts. 
In  an  invasion  of  pathogenic  germs  into  the  resisting 
organism  a  part  is  destroyed  by  substances  (alexins) 
already  present  in  the  serum  (and  derived  from  leu- 
cocytes);  another  part  is  destroyed  by  substances 
which  are  produced  from  leucocytes  (or  other  tis- 
sues) under  the  influence  of  the  bacteria.     A  part  of 


SUGAR   IK  THE    NUTRIENT  MEDIUM.  163 

the  germs  which  are  destroyed  by  the  leucocytes  is 
absorbed  by  the  latter  secondarily,  but  some  germs 
are  undoubtedly  ingested  alive  by  the  leucocytes. 
Metschnikoff  —  the  most  redoubtable  antagonist  of 
Buchner — insists  upon  the  view  that  the  latter  proc- 
ess (phagocytosis),  followed  by  subsequent  death  of 
the  germs  within  the  leucocytes,  is  the  essential  fea- 
ture of  natural  immunity. 

An  increase  of  the  congenital  resistance  to  various 
infectious  diseases  has  been  effected  in  a  number  of 
ways.  Thymus  extract,  spermin,  abrin  (toxic  al- 
buminoids from  the  paternoster  pea),  papayotin 
(albumin-dissolving  ferment  from  the  papaya),  cin- 
namic  acid,  iodine  trichloride,  sodium  carbonate,  etc., 
when  injected  into  animals  have  produced  favorable 
effects,  sometimes  in  one,  sometimes  in  several  infec- 
tious diseases.  Indeed,  an  increased  resistance  has 
been  observed  from  the  injection  under  the  skin,  but 
especially  into  the  peritoneal  cavity,  of  an  entire 
series  of  ordinary  albuminous  substances,  such  as 
blood  serum  and  bouillon. 

It  is  generally  assumed  that  this  effect  depends 
upon  increased  stimulation  of  the  leucocytes  to  the 
production  of  substances  which  are  antagonistic  to 
the  bacteria. 

According  to  the  majority  of  writers  there  is  a 
sharp  contrast  between  this  increased  resistance  and 
the  specific  immunity  from  a  definite  disease  which 
develops  when  an  individual  has  spontaneously  ac- 
quired and  passed  through  this  infectious  disease  or 
when  he  has  been  purposely  inoculated  with : 

(1)  Naturally  or  artificially  attenuated  infection- 
producers  of  the  same  variety ;  or 


164  ATLAS   OF   BACTERIOLOGY. 

(2)  Extinct  cultures  of  the  micro-organism  in  ques- 
tion; or 

(3)  The  blood  serum  or  tissue  juices  of  an  animal 
immunized  by  the  plans  mentioned  under  (1)  and  (2). 
After  (1)  and  (2)  there  develops  an  active  immunity  ^ 
after  (3)  a  passive  immunity. 

According  to  the  most  widely  entertained  opinion 
specific  immunity  depends  upon  the  presence  of 
specific  "  antisubstances"  (Behring)  in  the  blood  and 
tissues  of  the  immunized  animal.  According  to 
Buchner  the  "antisubstances"  are  derived  from  the 
injected  bacteria  cultures  and  are  much  more  resis- 
tant than  alexins  to  noxious  influences.  Thus 
tetanus  antitoxin  tolerates  a  temperature  of  70°-80° 
and  the  action  of  sunlight  and  putrefaction  without 
decomposing.  Brieger  and  Ehrlich  have  extracted 
diphtheria  antitoxin  in  a  solid  form  from  the  milk  of 
goats  which  were  rendered  immune  against  diph- 
theria. Whether  it  is  an  albuminoid  or  adheres  to  al- 
buminoids, is  not  yet  known.  The  antitoxins  are  best 
extracted  (Brieger  and  Boer:  Z.  H.,  XXI.,  266)  by 
means  of  zinc  chloride,  but  we  have  not  yet  succeeded 
in  freeing  them  from  the  last  traces  of  zinc.  Accord- 
ing to  Emmerich  the  "  antisubstances,"  which  he  calls 
"immune  proteidins,"  are  combinations  of  a  sub- 
stance furnished  by  the  bacteria  with  body  albumin 
from  the  immunized  animal. 

In  some  cases  the  character  of  immunity,  the  action 
of  the  "antisubstances,"  is  jjurely  antitoxic,  a  true 
antidote.  The  notion,  first  advanced  by  Behring  and 
Kitasato,  that  toxin  and  antitoxin  neutralize  one  an- 
other chemically  (somewhat  like  an  acid  and  its  base) 
has  not  been  corroborated.     We  have  to  deal  rather 


SUGAR  IK  THE   NUTRIENT  MEDIUM.  165 

with  an  antagonistic  action  upon  the  cells  of  the 
body  analogous  to  the  action  of  atropine  against 
morphine,  except  that  the  antisubstances  possess  a 
minimum  toxicity  or  none  at  all.  The  proof  that  an 
ineffective  mixture  of  toxin  and  antitoxin  still  contains 
a  virus  is  furnished,  for  example,  by  the  fact  that 
guinea-pigs,  upon  whom  antitoxin  has  less  protective 
action  than  upon  mice,  can  be  poisoned  with  mixtures 
of  toxin  and  antitoxin,  which  are  entirely  devoid  of 
effect  on  mice  (Buchner). 

While  the  "antisubstances"  of  diphtheria  protect 
very  well  against  the  diphtheria  virus,  they  have  no 
injurious  effect  on  the  diphtheria  bacilli  either  in 
vitro  or  in  vivo,  i.e.,  they  are  not  bactericidal.  The 
diphtheria  bacilli  may  grow  in  the  interior  of  an  im- 
munized organism  but  they  are  not  harmful. 

Entirely  different  in  principle  is  the  mode  of  action 
of  the  "antisubstances"  in  cholera.  Here  they  are 
exquisitely  bactericidal,  but  do  not  protect  against 
large  amounts  of  the  cholera  virus  (K.  Pfeiffer).  Ac- 
cording to  Emmerich,  this  is  also  true  of  hog  ery- 
sipelas and  pneumonia. 

Much  attention  has  been  devoted  to  the  question 
of  the  specific  action  of  the  "antisubstances."  Kich- 
ard  Pfeiffer,  the  strongest  advocate  of  their  absolutely 
specific  action,  has  defended  successfully  the  follow- 
ing standpoint  in  regard  to  the  cholera  vibrio  and  its 
allies :  Every  pathogenic  organism  furnishes,  in  the 
body  of  the  actively  immunized  animal,  "antisub- 
stances" which  exert  a  bactericidal  action  (often  ex- 
tremely pronounced)  only  against  the  organism  in 
question  but  not  against  its  closest  allies.  This  spe- 
cific action  is  so   pronounced  that  Pfeiffer  regards 


166  ATLAS   OF   BACTER10L0(^Y. 

it  as  the  most  valuable  diagnostic  measure,  for  ex- 
ample, in  deciding  the  question  whether  an  organism 
is  to  be  regarded  as  a  cholera  vibrio  or  not.  Pfeiffer 
made  the  same  discovery  in  regard  to  bacterium  typhi 
and  its  allies,  and  this  is  corroborated  by  Dunbar, 
Sobernheim,  Loffler,  and  Abel. 

It  must  not  be  forgotten,  however,  in  opposition  to 
these  very  interesting  and  surprising  findings  that  a 
number  of  investigators  (for  example,  Hiippe)  do 
not  recognize  a  sharp  distinction  between  resistance 
and  specific  immunity,  but  acknowledge  only  quanti- 
tative, not  qualitative,  differences.  At  all  events,  we 
still  have  much  to  learn  in  this  difficult  field. 

Technical  Appendix. 

The  following  recommendations  and  brief  descrip- 
tions furnish  all  the  technical  directions  which  are 
given  in  a  thorough  course  of  bacteriology.  We  have 
given  only  the  most  necessary  data  and  those  which 
in  our  experience  have  proved  most  practical. 

I.    MICROSCOPICAL  EXAMINATION   OF  BACTERIA. 

1.  Hints  on  Microscopical  Technique. 

For  bacteriological  examinations  we  use  almost  ex- 
clusively the  modern  microscope  with  Abbe's  illu- 
minating apparatus,  iris  diaphragm,  a  low-power 
lens,  and  an  oil  immersion  lens. 

A.  Low  magnifying  power  (sixty  to  one  hundred 

times)  and  narrow  diaphragm  are  used  for  careful 

examination  of  plate  cultures.     For  this  purpose  we 

either  raise  the  cover*  and  examine  the  colony  from 

*Our  plate  cultures  are  always  poured  into  cups. 


TECHNICAL   APPENDIX.  167 

above,  or,  if  we  do  not  wish  to  soil  the  plate  by  open- 
ing it,  place  it  upon  the  cover  and  examine  the  colony 
from  below.  This  does  not  give  such  characteristic 
appearances  in  all  cases. 

B.  High  magnifying  power.  Oil  immersion  (seven 
hundred  to  twelve  hundred  times)  is  used  in  the  ob- 
servation of  individual  bacteria.  Upon  the  prepara- 
tion is  placed  a  drop  of  oil  of  cedar,  the  tube  of  the 
microscope  pushed  down  by  means  of  the  coarse  ad- 
justment until  the  lens  just  touches  the  surface  of  the 
oil,  and  then  adjust  it  accurately  on  the  preparation 
with  the  micrometer  screw. 

(a)  Unstained  Preparations.  Narrow  diaphragm. 
They  are  examined  in  two  ways : 

1.  A  drop  of  a  fluid  pure  culture  or  a  drop  of  water 
mixed  with  a  trace  of  pure  culture  is  placed  between 
the  slide  and  cover-glass ;  or 

2.  In  the  hanging  drop.  A  platinum  loopful  of 
fluid  pure  culture,  or  a  loopful  of  bouillon  mixed  with 
a  trace  of  pure  culture,  is  placed 
on  a  cover-glass,  and  this  laid 
(reversed)  upon  a  slide  which 
has  been  hollowed  out  so  that  the  drop  lies  in  the 
cavity.  The  cover-glass  is  then  fixed  to  the  slide 
by  applying  a  trace  of  water  to  the  four  corners  of 
the  cover-glass  or  by  apjilying  vaseline,  if  prolonged 
observation  is  required. 

(6)  Stained  Preparations.  Open  diaphragm. 
Abbe's  illuminating  apparatus.  To  observe  double- 
stained  section  preparations  we  require  wide  dia- 
phragm for  the  bacteria  and  narrow  diaphragm  for 
the  tissues. 

C.  Cleansing  of  the  preparations  and  the  micro- 


168  ATLAS   OF   BACTERIOLOGY. 

scope.  The  immersion  oil  is  always  brushed  off 
gently,  and  now  and  then  the  lens  is  rapidly  cleansed 
with  xylol  and  chamois  skin;  prolonged  action  of 
xylol  loosens  the  setting  of  the  lens.  Xylol  also  re- 
moves dried  particles  of  oil  from  the  cover-glasses  of 
old  preparations. 

2.  The  Most  Important  Solutions  for  Making 
Preparations. 

A.  Staining  Solutions. 

1.  Watery  alcoholic  solution  of  fuchsin  and  methyl 
blue.  A  concentrated  "stock  solution"  is  made  by 
pouring  absolute  alcohol  over  the  powdered  coloring 
matters  (fuchsin,  methyl  blue)  in  bottles,  shakiug, 
letting  them  stand  for  a  few  hours,  and  then  filtering. 
Of  this  saturated  solution  one  part  is  mixed  with  four 
parts  distilled  water  and  filtered  before  using.  In 
order  to  obtain  good  preparations  it  is  better  to  stain 
for  a  longer  time  with  weak  solutions  than  for  a 
shorter  time  with  strong  solutions. 

2.  Carbolized  fuchsin  (Ziehl's  solution) : 

Fuchsin 1.0  gm. 

Acid,  carbolic,  liq 5.0    " 

Alcohol 10.0    " 

Aq.  dest 90.0    " 

3.  Aniliue  fuchsin:  4.0  aniline  oil  (anilin.  pur.)  are 
well  shakeu  for  several  minutes  with  100  aq.  dest., 
then  filtered  until  all  the  water  runs  off  clear  (then 
the  funnel  is  removed  because  otherwise  the  oil  will 
pass  through).  In  this  aniline  water  are  dissolved  4.0 
gm.  fuchsin  and  it  is  then  again  filtered. 


TECHNICAL   APPENDIX.  169 

4.  Aniline  gentian  (Elirlich's  solution):  To  100 
c.c.  aniline  water  add  11  c.c.  of  an  alcoholic  con- 
centrated gentian  violet  solution  (stock  solution). 
This  solution  does  not  keep  long. 

5.  Loffler's  methyl  blue:  To  100  c.c.  water,  which 
contains  1  c.c.  of  a  one-per-cent  potash  lye,  add 
30  c.c.  of  a  concentrated  alcoholic  solution  of  methyl 
blue.  The  staining  power  is  increased  by  the  addi- 
tion of  the  alkali. 

6.  Bismarck  brown:  Prepare  like  No.  1.  (Stains 
tissues,  but  bacteria  poorly). 

7.  Alum  carmine:  To  100  c.c.  of  a  five-per-cent 
alum  solution  add  2  gm.  carmine,  boil  for  an  hour, 
and  filter. 

B.  Differentiation  Measures. 

1.  Distilled  water. 

2.  Absolute  alcohol. 

3.  Iodine-potassium  iodide  solution  (Gram). 

lodin.  pur  .    1.0 

Potassii  iodidi 3.0 

Aq.  destil 300.0 

4.  Sulphuric  acid  (twenty-five  per  cent). 

5.  Acetic  acid  (three  per  cent). 

6.  Acid  alcohol. 

Alcohol  (ninety  per  cent) 100  c.c. 

Distilled  water. 200   " 

Pure  hydrochloric  acid 20  gtt 

G.  Mordants  for  the  Fhgella. 
Loffler's  mordant: 
10  c.c.  alcoholic  solution  of  fuchsin. 
50  c.c.  cold  saturated  ferrosulphate  solution. 
100  c.c.  twenty -per-cent  tannin  solution. 


170  ATLAS   OF   BACTERIOLOGY. 

2.  Bunge's  mordant: 

25  c.c.  of  a  twentyfold  diluted  officinal  ferric  chloride 

solution. 
75  c.c.  saturated  watery  solution  of  tannin. 

To  this  solution  is  added,  immediately  before  using, 
enough  of  a  tliree-per-cent  solution  of  hydrogen  per- 
oxide to  produce  a  reddish-brown  color,  and  it  is 
then  filtered  (we  have  always  dispensed  with  the 
peroxide). 

D.  Substances   Used  for  Clearing  Up  and  Mounting. 

1.  Xylol. 

2.  Canada  balsam. 

3.  Dammar  varnish. 

3.  Preparation  of  Stained  Specimens  of  Bacteria. 
A.  Smear  Preparations. 

1.  Ordinary  Stain  with  Fuchsin  or  Methyl  Blue. 
This  may  be  used  for  all  bacteria  with  the  exception 
of  the  tubercle  bacillus. 

We  place  upon  the  cover-glass  or  slide  a  loopful  of 
distilled  water,  mix  with  it  a  trace  of  pure  culture 
(best  from  a  solid  nutrient  medium)  and  then  spread 
the  drop  in  a  very  thin  layer.  After  the  fluid  has 
evaporated  the  preparation,  with  the  layer  turned  up, 
is  rapidly  drawn  three  times  through  the  flame  in 
order  to  fni  the  bacteria  on  the  glass  (not  to  burn 
them)  and  the  layer  of  bacteria  is  covered  with  the 
staining  solution.  After  a  brief  interval  (one  minute), 
perhaps  after  feebly  warming  the  glass,  the  prepara- 
tion is  washed  with  water  and  allowed  to  dry  (some- 


TECHNICAL  APPENDIX.  171 

times  after  cautious  warmiug).     By  means  of  a  drop 
of  Canada  balsam  the  dry  cover-glass  is  finally  fixed 
to  the  slide  with  the  bacterial  layer  downward. 
2.  Gram's  Stain. 

(1)  Making  the  smear  preparation  as  above. 

(2)  Staining  with  Ehrlich's  solution  three  minutes. 

(3)  Washing  off  with  water. 

(4)  Differentiation  with  iodine-potassium  iodide  so- 
lution one  minute. 

(5)  Decolorizing  with  absolute  alcohol  up  to  color- 
lessness  (usually  one  to  two  minutes). 

(6)  Drying  and  mounting. 

For  the  species  which  are  adapted  to  Gram's  stain, 
vide  the  table.  In  our  experience  the  common 
opinion  that  every  variety  of  bacteria  may  be  pre- 
pared invariably  either  well  or  not  at  all  according 
to  this  method  is  erroneous.  For  example,  we  ob- 
served among  the  fluorescents,  which  are  usually 
described  in  literature  as  unstainable,  that  three 
varieties  out  of  twelve  stained  very  beautifully  after 
twenty -four  hours'  culture.  Indeed,  according  to 
Zimmermann,  all  fluorescents  may  be  stained  in 
young  cultures. 

In  like  manner  we  were  able  to  stain  the  bacillus 
of  symptomatic  anthrax  which  has  often  been  re- 
garded as  incapable  of  staining.  The  contradictory 
statements  may  be  explained  in  part  by  the  fact  that 
the  material  emi3loyed  has  varied  greatly  in  age,  and 
also  that  the  differentiation  with  alcohol  was  effected 
in  different  ways.  But  tyrothrix  tenuis,  which  has 
been  regarded  as  unstainable  by  Gram's  method,  was 
found  to  stain  very  well  on  a  subsequent  test  of  the 
same  culture  with  the  same  technique.     At  all  events 


172  ATLAS   OF   BACTERIOLOGY. 

at  each  staining  a  fresh  preparation  of  anthrax  bacil- 
lus should  be  stained  at  the  same  time  and  all  prepa- 
rations differentiated  with  alcohol  for  an  equally  long 
time  (one  or  two  minutes).  We  can  then  judge  very 
well  whether  one  variety  of  bacteria  retains  or  gives 
off  the  coloring  matter. 

3.  Capsule  Preparation.  According  to  Johne  we 
proceed  in  the  following  manner : 

(1)  Heating  the  preparation  with  two-per-cent  so- 
lution of  gentian  violet  until  steam  is  given  off.  ^ 

(2)  Washing  with  water. 

(3)  Moistening  with  two-per-cent  acetic  acid  for 
six  to  ten  seconds. 

(4)  Washing  with  water. 

By  this  method  a  very  distinct  membrane  around 
the  intensely  colored  bacterium  cell  can  often  be 
demonstrated  in  varieties  which  are  not  regarded  as 
"capsular  bacteria."  The  capsules  are  seen  best  on 
examination  in  water. 

4.  Staining  of  Flagella.  The  flagella,  which  are 
almost  always  invisible  when  unstained,  are  generally 
prepared  according  to  LofBer's  method : 

(1)  Rubbing  up  a  trace  of  young  agar  streak  cul- 
ture (not  bouillon)  in  a  very  small  drop  of  water; 
spread  out  well,  dry  rapidly. 

(2)  Heating  of  the  preparation  with  mordant  until 
steam  is  produced  (do  not  boil)  for  one-half  to  one 
minute. 

(3)  Washing  off  in  a  vigorous  stream  of  water. 

(4)  Washing  off  in  alcohol  in  order  to  remove  the 
remains  of  the  mordant  adherent  at  the  edges. 

(5)  Dropping  of  the  staining  fluid  (a  fev/  crystals 
are  dissolved  in  10  c.c.  aniline  water,  and  then  one 


TECHNICAL   APPENDIX.  173 

per  cent  soda  lye  is  added  drop  by  drop  until  the 
clear  fluid  just  begins  to  grow  opaque)  and  beating 
for  one  minute  until  steam  is  evolved. 

(6)  Washing  off  in  water,  drying,  mounting  in 
Canada  balsam. 

The  manipulations  must  be  carried  out  with  the 
most  scrupulous  cleanliness,  and  the  cover-glasses 
must  be  especially  well  cleaned  with  acids  and  alco- 
hol. The  cultures  must  be  young,  although  it  is  not 
necessary,  as  some  authors  maintain,  to  make  the 
staining  only  in  cultures  that  are  twenty-four  hours 
old.  We  have  often  obtained  very  good  preparations 
even  at  the  end  of  twelve  days.  The  mordants  are 
usually  prepared  fresh. 

According  to  Loffler,  it  is  necessary,  in  the  case  of 
the  majority  of  bacteria,  to  add  a  definite  amount  of 
acid  or  alkali  to  the  mordant  in  order  to  obtain  well- 
stained  flagella.  Loffler  advises  that  to  16  c.c.  of  the 
mordant  there  be  added  for : 

Drops.  Soda  lye. 

Cholera  vibrios i  to    1  1  per  cent 

Spirillum  rubrum 9  1 

Bacterium  typhi 20  to  22  1 

Bacillus  subtilis 28  to  38  1 

Bacillus  oedematis  maligni. ..  36  to  37  1 

Bacterium  pyocyaneum 5  to    6  Equivalent  sul- 
phuric acid. 

Oar  results  show  that  in  the  majority  of  cases  we 
obtain  very  useful  pictures  with  the  unchanged  mor- 
dant and  that  the  addition  of  alkali  or  acid  is  by  no 
means  material.  Similar  experiences  have  been  had 
by  other  writers,  for  example,  Lucksch,  Giinther,  A. 
Fischer,  Nicolle  and  Morax,  but  our  investigations 
have  not  been  concluded. 


174  ATLAS   OF    BACTERIOLOGY. 

Bunge  lias  recently  employed  a  somewhat  different 
method  which  also  gave  us  good  results,  but,  like 
Loffler's  method,  occasionally  left  us  capriciously  in 
the  lurch. 

(1)  Preparation  of  the  specimen,  according  to 
Loffler. 

(2)  Heating  with  Bunge's  mordant  for  one  minute 
until  steam  is  produced. 

(3)  Careful  cleaning  with  water  and  drying. 

(4)  Warming  slightly  with  carbolized  gentian  violet 
or  carbolized  fuchsin. 

(5)  Washing  in  water,  drying,  and  mounting  in 
Canada  balsam. 

Most  of  our  specimens  are  i)repared  with  Bunge's 
mordant  which  is  several  months  old. 
5.  Staining  of  Endospores.* 
According  to  Hauser : 

(1)  Preparation  of  the  specimen.  (It  should  be 
drawn  ten  times,  instead  of  three  times,  rapidly 
through  the  flame.) 

(2)  Staining  with  watery  fuchsin  or  carbolized 
fuchsin  (Ziehl's  solution) ;  the  preparation,  over  the 
flame,  is  covered  freely  with  the  staining  fluid,  and 
heated  (not  boiled)  one  to  two  minutes  until  there  is 
an  indication  of  simmering.  The  evaporating  stain- 
ing fluid  is  replaced  constantly  by  fresh  fluid. 

(3)  Washing  with  acid  alcohol,  f  until  the  red  color 
of  the  preparation  is  almost  gone. 

*  Arthrospores  possess  no  undisputed  color  reactions.  For 
metacJiromatic  corpuscles,  Ernst's  and  Bunge's  granules,  pre- 
liminary stages  of  spores,  mde  page  71. 

f  Instead  of  acid  alcohol  we  may  also  use  thirty  per  cent 
nitric  acid,  five  or  twenty  five  per  cent  sulphuric  acid,  but 
these  must  be  allowed  to  act  for  a  shorter  period. 


TECHNICAL   APPEI^DIX.  175 

(4)  After-staining  with  methyl  blue  (a  few  sec- 
onds).    The  spores  remain  red,  the  bacilli  blue. 

6.  Staining  of  Tubercle  Bacilli.  This  is  done  ac- 
cording to  the  same  principles  as  the  staining  of 
spores.  The  preparation  is  treated  in  the  flame 
with  a  deeply  staining  solution  and  then  everything 
with  the  exception  of  the  tubercle  bacilli  is  decolor- 
ized with  some  acid  solution. 

(a)  We  may  manipulate  exactly  as  in  spore  staining 
(according  to  Ziehl-Neelsen),  except  that  the  prepara- 
tion is  drawn  only  three  times  through  the  flame. 
This  method  is  the  only  one  employed  by  us.  An- 
other favorite  method  is  the  one  recommended  by  A. 
Fraenkel  and  Gabbet,  in  which  decolorization  and 
after-staining  are  effected  at  the  same  time.  Then 
the  preparation  which  has  been  stained  with  hot  car- 
bolized  fuchsin,  and  washed  in  water,  is  placed  in  the 
following  solution: 

Sulphuric  acid 1 

Distilled  water 3 

Methyl  blue,  q.s.  uutil  the  most  intense  blue  color  is  pro- 
duced. 

We  then  wash  carefully  in  water,  dry,  and  mount  in 
Canada  balsam. 

However  convenient  this  method  may  be,  it  is 
better,  for  those  who  are  not  very  experienced,  to 
stain,  differentiate  with  acids,  and  after-stain  sepa- 
rately, because  in  this  way  success  is  more  assured. 

(h)  Ehrlich-Koch's  method  is  also  often  employed. 
The  dry  preparation  is  drawn  through  the  flame, 
treated  with  aniline  gentian  solution  for  one  to  two 
minutes  over  the  flame  and  heated  with  acid  (usually 
thirty  per  cent  nitric  acid)  for  one  to  four  seconds, 


176  ATLAS   OF   BACTERIOLOGY. 

and  with  sixty  per  cent  alcohol  for  a  few  moments. 
It  is  then  dipped  for  several  minutes  in  a  watery 
solution  of  Bismarck  brown  and  washed  off  in  water. 
The  tubercle  bacilli  then  appear  violet  on  a  brown 
background. 

In  this  form  the  method  is  suitable  for  cover-glass 
preparations  from  pure  cultures  and  tuberculous 
sputum  with  many  tubercle  bacilli.  If  very  few  or  no 
bacilli  are  found  in  the  first  preparations,  we  must 
adopt  some  method  for  increasing  their  numbers. 
We  mention  two  of  the  innumerable  recommen- 
dations : 

{a)  According  to  Strohschein : 

Five  to  ten  cubic  centimetres  of  the  sputum  are 
mixed  with  a  threefold  amount  of  Wendriner's  borax- 
boracic  acid  solution,*  and  after  vigorous  shaking 
allowed  to  settle  for  four  to  five  days.  The  mixture 
becomes  fluid  and  the  bacilli  settle  at  the  bottom. 
Such  sputum  may  be  used  for  examination  even  after 
the  lapse  of  years. 

{h)  According  to  Dahmen,  modified  by  Heim : 

The  entire  sputum  is  cooked  from  fifteen  to  twenty 
minutes  in  a  beaker  glass  in  the  steam  chamber,  then 
allowed  to  cool,  the  opalescent  fluid  is  poured  off,  and 
the  crumbly  sediment  is  used  for  smear  preparations. 

B.  Section  Preparations. 

1.  Universal  method,  according  to  Loffler,  adapted 
to  the  large  majority  of  bacteria. 
The  section,  which  lies   in  alcohol,  is   conveyed 

*  Eight  grams  borax  dissolved  in  hot  water,  12  gin.  horacic 
acid  added,  and  then  4  gr.  borax ;  after  crystallization  the  solu- 
tion is  filtered. 


TECHNICAL   APPENDIX.  177 

(spread  upon  a  spatula  of  German  silver  or  glass)  to 
Loffler's  alkaline  methyl  blue  solution  for  from  five  to 
thirty  minutes,  and  is  then  placed  for  a  few  seconds 
in  one-per-cent  acetic  acid.  After  the  differentiation 
the  section  is  placed  in  absolute  alcohol,  xylol,  and 
Canada  balsam.  We  must  try  how  long  the  acetic 
acid  may  be  allowed  to  act,  and  must  accelerate  the 
dehydration  in  alcohol  as  much  as  possible;  the 
bacilli  should  be  blackish-blue,  the  nuclei  blue,  the 
protoplasm  bluish. 

2.  Nicolle  states  that  by  the  following  method  he 
has  obtained  very  good  section  staining  of  objects 
which  are  stained  with  difficulty — for  example,  in 
glanders,  typhoid  fever,  etc. : 

Loffler's  blue,    one  to  three  minutes. 

Washing  in  water. 

Treatment  with  ten-per-cent  solution  of  tannin  for 
a  few  seconds. 

Washing  in  water. 

Absolute  alcohol,  oil  of  cloves,  xylol,  Canada 
balsam. 

3.  According  to  Gram : 

(1)  Ehrlich's  solution,  three  minutes. 

(2)  Iodine-potassium  iodide  solution,  two  minutes. 

(3)  Alcohol,  one-half  minute. 

(4)  Alcohol  containing  three  per  cent  hydrochloric 
acid,  ten  seconds. 

(5)  Alcohol,  several  minutes  until  maximum  decol- 
orization. 

(6)  Xylol;  finally  mounting  in  Canada  balsam. 

If  the  tissues  are  to  be  stained  in  a  contrasting 
color,  the  section  is  placed,  after  the  maximum  de- 
colorization    with     alcohol,    in    a    watery    solution 
1^ 


178  ATLAS   OF    BACTERIOLOGY. 

(10  :  100)  of  Bismarck  brown  for  a  few  minutes,  then 
in  absolute  alcohol  for  fifteen  to  twenty  seconds,  then 
in  xylol,  and  finally  in  Canada  balsam. 

4.  Botkin  maintains  that  Gram's  stain  is  facilitated 
by  washing  in  aniline  water  preparations  which  have 
been  stained  with  aniline  gentian.  The  preparations, 
when  taken  from  the  iodine  solution,  subsequently 
stand  the  action  of  the  alcohol  very  much  better. 
Bacillus  oedematis  maligni  and  bacterium  pneu- 
moniae Friedlander  can  be  stained  in  this  way. 

5.  Kutscher's  modification  of  Gram's  method: 

A  concentrated  solution  of  gentian  violet  is  made 
in  a  mixture  of : 

Aniline  water 1  part. 

Alcohol 1     *' 

Five-per-cent  carbolized  water 1     " 

This  concentrated  solution  is  poured  drop  by  drop 
into  a  watch-glassful  of  water  until  a  shimmering 
layer  forms  on  the  surface.  The  sections  are  placed 
in  this  for  ten  to  fifteen  minutes,  are  then  washed  off 
in  distilled  water,  placed  one  minute  in  iodine- 
potassium  iodide,  then  in  alcohol,  xylol,  and  bal- 
sam. Malignant  oedema  and  symptomatic  anthrax 
can  also  be  stained  by  this  method. 

6.  If  tubercle  bacilli  are  to  be  stained  in  sections 
we  use  carbolized  fuchsin  or  aniline  gentian  solution 
as  in  cover-glass  staining,  but  we  dispense  with  the 
heating  and  instead  allow  the  staining  fluid  to  act  for 
fifteen  to  thirty  minutes, 


TECHNICAL  APPENDIX.  179 


4.  Production  of  Section  Preparations. 

At  the  autopsy  small  pieces  of  the  organs  are 
thrown  at  once  into  an  abundance  of  absolute  alcohol 
and  kept  there  two  to  three  days,  the  alcohol  being 
renewed  two  to  three  times.  In  most  cases  the 
organs  are  then  ready  for  cutting.  For  this  pur- 
pose the  firmer  part  of  the  kidneys,  liver,  and  muscles 
are  placed  on  a  piece  of  cork  with  liquefied  commer- 
cial gelatin  *  and  then  again  placed,  with  the  cork,  in 
absolute  alcohol.  At  the  end  of  twenty -four  hours  the 
organ  may  be  cut  with  the  microtome.  More  delicate 
organs  must  be  embedded  in  celloidin  or  paraffin ;  be- 
fore staining,  the  paraffin  is  removed  completely  by 
washing  repeatedly  in  turpentine,  or  xylol  and  the 
prei^aration  is  placed  in  absolute  alcohol  after  re- 
moval from  the  xylol. 

II.   CULTURE  OF  BACTERIA. 

1.  Nutrient  Media. 

A.  Non-albuminous  (according  to  C.  Fraenkel  and 

Voges). 

Common  salt 5  gm. 

Neutral  commercial  sodium  phosphate 2    " 

Ammonium  lactate 6    " 

Asparagin 4    " 

are  dissolved  in  1,000  gm.  of  distilled  water.     We  may 

add  ten  per  cent  gelatin  or  one  per  cent  agar,  and 

thus  obtain  a  non-saccharine  nutrient  medium  which 

*  One  part  of  gelatin  is  dissolved  in  two  parts  of  water. 


180  ATLAS    OF    BACTERIOLOGY. 

is  suitable  to  the  majority  of  bacteria.     The  addition 
of  milk  sugar  gives  a  milk-sugar  uutrient  medium 
which  is  free  from  dextrose  (Lehmann  and  Neumann). 
B.  Albuminous. 

1.  Peptone  water.  In  1  litre  of  water  are  dissolved 
10  gm.  dried  peptone,  and  5  gm.  sodium  chloride,  and 
sterilized  together. 

2.  Milk.  Fresh  milk  (best,  fresh  centrifugal  milk) 
is  placed  in  test  tubes  and  sterilized  in  the  steam 
chamber  for  one-half  hour  on  two  successive  days. 
Milk  which  contains  the  spores  of  the  subtilis  group 
is  often  incapable  of  sterilization. 

3.  Litmus  whey  (Petruschky).  Casein  is  cau- 
tiously precipitated  from  milk  by  giving  it  a  very 
feeble  acid  reaction  with  diluted  hj-^drochloric  acid, 
the  filtrate  is  boilefl  and  filtered,  and  the  neutralized 
fluid  mixed  with  some  litmus.  This  whey  is  not 
easily  prepared  {vide  Heim:  "Lehrbuch,"  p.  210). 

4.  Hay  decoction.  About  10  gm.  dry  hay  are  boiled 
in  a  litre  of  water.  The  filtered  solution  is  placed  in 
test  tubes,  and  sterilized  for  two  hours  on  three  suc- 
cessive days  (kept  over  night  in  the  incubating  cham- 
ber) in  order  to  destroy  the  very  resisting  spores. 

5.  Beer  wort  (not  neutralized)  is  allowed,  after 
sterilization,  to  settle  for  a  few  weeks,  then  poured 
off  clear  into  test  tubes,  and  again  sterilized. 

6.  Nutrient  bouillon. 

(a)  From  meat :  500  gm.  lean  beef  are  boiled  upon 
the  flame  for  one-half  hour  with  1,000  gm.  of  water  in 
an  enamelled  pot,  filtered,  the  filtrate  reduced  to  1,000 
gm.  and  10  gm.  peptone  with  5  gm.  sodium  chloride 
added;  this  is  placed  in  the  steam  chamber  until 
dissolved,   and  the  whole   is   then  neutralized  with 


TECHNICAL  APPENDIX.  181 

normal  soda  lye  (indicator,  phenolphthalein).*    We 
then  filter,  pour  into  test  tubes,  and  sterilize. 

(h)  From  meat  extract :  10  gm.  meat  extract  are  dis- 
solved in  1,000  gm.  water,  5  gm.  sodium  chloride  and 
10  gm.  peptone  are  added,  the  solution  is  neutralized 
and  well  sterilized  several  times. 

7.  Potato  water  for  tubercle  bacilli :  500  gm.  peeled 
potatoes  are  rubbed  upon  a  grater,  allowed  to  remain 
over  night  in  500  gm.  water  in  the  refrigerator,  de- 
canted, filled  up  to  1000  gm.,  cooked  for  an  hour  in  the 
water-bath,  filtered,  four  per  cent  glycerin  is  added, 
and  the  mixture  sterilized. 

8.  Gelatin  nutrient  media. 

(a)  Meat  water-peptone  gelatin  (ordinary  "gela- 
tin" or  "nutrient  gelatin"  of  the  laboratories). 

To  1,000  gm.  bouillon  (vide  nutrient  bouillon)  are 
added  100  gm.  gelatin,  10  gm.  peptone,  5  gm.  sodium 
chloride,  the  mixture  is  heated  in  the  steam  chamber 
until  all  the  ingredients  are  liquefied,  neutralized 
with  normal  soda  lye,  sterilized,  and  filtered.  After 
the  melted  gelatin  is  placed  in  test  tubes  it  is  again 
sterilized. 

(b)  Meat- water  gelatin:  the  same  as  under  (a),  but 
without  peptone  and  sodium  chloride. 

(c)  Beer-wort  gelatin  is  made  by  adding  ten  per 
cent  gelatin  to  the  wort ;  it  should  not  be  neutralized. 

(d)  Plum-decoction  gelatin :  500  gm.  dried  plums 
are  cooked  in  500  gm.  water,  the  fluid  is  poured  off, 
and  the  plums  are  again  cooked  with  500  gm.  water. 

*  Illustration  :  Ten  cubic  centimetres  bouillon  require  for  sat- 
uration 2.2C.C.  one-tonfih  normal  soda  lye;  1,000  c.c.  bouillon 
require  for  saturation  220  c.c.  one-tenth  normal  soda  lye,  or  22 
c.  c.  normal  soda  lye. 


182  ATLAS   OF   BACTERIOLOGY. 

Both  fluids  are  then  mixed,  filtered,  and  ten  per  cent 
gelatin  is  added.     Not  to  be  neutralized. 

(e)  Herring  gelatin.  Two  salt  herring,  unwashed, 
are  boiled  in  1,000  gm.  water  and  ten  per  cent  gelatin 
is  added  to  the  filtrate ;  not  to  be  neutralized. 

(/)  Potato-water  gelatin,  according  to  Holz,  for 
bacterium  typhi:  500  gm.  potatoes  are  thoroughly 
washed,  peeled,  finely  grated,  and  squeezed  through 
a  linen  cloth.  The  opaque  juice  may  be  allowed  to 
settle  for  twenty -four  hours  and  then  filtered,  or,  as 
we  always  prefer,  filtered  at  once  through  pure 
animal  charcoal.  After  heating  one  hour  in  the 
steam  chamber  ten  per  cent  gelatin  is  added  to  the 
clear  fluid,  this  is  again  heated  in  the  steam  chamber, 
filtered,  poured  into  test  tubes  and  sterilized  on  three 
successive  days. 

(g)  Potassium  iodide  potato-water  gelatin  (Eisner) : 
One  per  cent  iodide  is  added  to  the  gelatin.  The 
best  way  is  to  add  a  well-sterilized  solution  in  the 
requisite  amounts  to  gelatin  which  has  just  been 
made  ready  for  use. 

9.  Nutrient  agar.  To  1,000  gm.  bouillon  add 
10  gm.  very  finely  divided  agar,  boil  for  one  hour 
on  the  fire  in  a  glass  retort  until  completely  dis- 
solved; the  water  which  has  evaporated  is  replaced 
and  then  10  gm.  peptone  and  5  gm.  sodium  chloride 
are  added.  After  heating  again  in  the  steam  cham- 
ber the  fluid  is  neutralized,  filtered  by  means  of  the 
hot-water  funnel,  placed  in  test  tubes,  and  again 
sterilized. 

10.  In  order  to  make  grape-sugar  or  milk-sugar 
agar,  two  per  cent  of  the  corresponding  substance  is 
added  with  the  peptone  and  sodium  chloride.     As 


TECHNICAL   APPENDIX.  183 

bouillon  agar  generally  contains  traces  of  grape 
sugar,  we  have  for  some  time  made  a  milk-sugar 
agar  which  is  free  from  grape  sugar,  according  to  the 
plan  described  under  A. 

11.  Glycerin  agar.  To  the  nutrient  agar  is  added 
five  per  cent  glycerin,  the  mixture  poured  into  test 
tubes  and  sterilized. 

12.  Sugar-chalk  agar.  Mix  melted  sugar  agar 
with  finely  powdered,  dry,  sterilized  carbonate  of 
lime  until  the  mixture  becomes  cloudy  and  opaque, 
inoculate  the  bacteria  into  it,  and  pour  out  in  plates. 

13.  Potatoes.  After  careful  washing  the  potatoes 
are  peeled,  cut  into  discs  1  cm.  thick,  and  sterilized 
several  times  in  high  Petri's  dishes.  We  may  also 
perforate  the  peeled  potato  with  a  large  cork  borer 
and  divide  \lie  cylinder  by  an  oblique  cut  into  two 
wedges.  The  pieces  are  then  placed  in  a  test  tube 
at  the  bottom  of  which  is  a  little  dry  cotton  (to  ab- 
sorb the  water  ci  condensation)  and  sterilized  several 
times  in  the  steam  chamber. 

14.  Blood  serum.  The  blood,  taken  from  the 
slaughtered  animal  under  proper  precautions,  is  al- 
lowed to  stand  for  twenty -four  hours  in  well  cleaned 
glass  cylinders  in  the  refrigerator;  on  the  following 
day  the  serum  is  removed  by  means  of  large  sterile 
pipettes.  It  is  placed  in  bottles,  one  per  cent  chloro- 
form is  added,  and  is  then  allowed  to  stand  for  a  few 
weeks,  being  shaken  occasionally.  For  use,  we  place 
the  serum,  which  has  been  poured  into  tubes,  in  the 
incubating  chamber  for  a  few  days  in  order  that  the 
chloroform  may  escape  completely.  It  is  employed 
either  in  the  fluid  state  or  after  it  has  been  made  rigid 
at  a  temperature  of  65°. 

% 


184  ATLAS   or   BACTERIOLOGY. 

15.  Loffler's  serum  mixture  for  diphtheria  bacilli. 
Three  parts  of  beef  or  sheep  serum  are  mixed  with 
one  part  calf's  bouillon,  which  contains  one  per  cent 
grape  sugar,  one  per  cent  peptone,  and  one-half  per 
cent  sodium  chloride. 

16.  Entirely  different  from  the  other  media  is  that 
first  devised  by  Kiihne,  modified  by  various  writers, 
and  finally  made  somewhat  more  practicable  by 
Stutzer  and  Burri.  We  refer  to  the  silicic  acid  nu- 
trient medium.  Gelatinous  silicic  acid,  whieh  is 
merely  mixed  with  a  few  salts,  is  an  important  nu- 
trient medium  for  certain  organisms  (for  example,  the 
nitrate-producers)  on  account  of  the  lack  of  organic 
nutrient  substances.  For  the  somewhat  complicated 
manipulation,  vide  Stutzer  and  Burri  (C.  B.,  Yol.  I., 
Part  v.,  722). 

2.  The  Employment  of  the  Duterent  Nutrient 
Media  Depends  upon  the  Following  View- 
Points  : 

I.  Fluids  (bouillon,  sugar  bouillon,  milk,  non- 
albuminous  nutrient  solution). 

1.  To  produce  cultures  en  masse. 

2.  To  obtain  bacterial  solutions  containing  an  ac- 
curately determinable  number  of  bacteria  (counting 
by  means  of  plates). 

3.  To  observe  the  development  of  membrane  and 
sediment. 

4.  To  study  the  metabolic  products. 
II.  Solid  Nutrient  Media. 

1.  Gelatinous  nutrient  media.  The  most  exten- 
sive use  is  made  of  gelatinous,  transparent  nutrient 


TECHNICAL   APPENDIX.  186 

media  (agar  and  gelatin)  and  for  the  following  rea- 
sons : 

(a)  They  may  be  used  as  fluids  and  as  solid  media : 
as  fluids  they  permit  the  separation,  as  solid  sub- 
stances the  fixation,  of  the  isolated  germs  and  their 
separate  growth  into  colonies. 

(b)  On  account  of  their  transparency  they  permit  a 
macroscopic  as  well  as  a  microscopic  observation  of 
the  cultures;  they  permit  a  differential  diagnosis  of 
the  varieties  and  an  early  recognition  of  any  im- 
purities. 

They  are  used  particularly : 

(a)  For  plate  cultures,  *.e.,  as  a  proof  of  positive 
separation  and  for  the  enumeration  of  individuals  and 
varieties. 

(b)  To  secure  characteristic  macroscopic  cultures, 
which  will  serve  for  differential  diagnosis. 

(c)  For  permanent  cultures  or  collections  of  living 
bacteria. 

The  special  advantages  of  agar  and  gelatin  are : 

(a)  Gelatin.  Advantages :  Easily  produced,  easily 
formed  into  plates  (at  25°) ;  its  property  of  liquefac- 
tion by  certain  bacteria  possesses  great  diagnostic 
importance.  Disadvantages :  As  it  melts  at  25°,  it 
cannot  be  used  in  hot  weather  and  at  incubating  tem- 
perature. 

(b)  Agar.  Advantages :  Practicable  at  incubating 
temperature  (i.e.,  for  the  rapid  culture  of  bacteria 
[spores]  and  particularly  of  thermophile  bacteria). 
Disadvantages:  Difficulty  of  preparation;  not  so 
easily  formed  into  plates.  The  cultures  are  often  not 
very  characteristic. 

2.  Blood  serum  and  glycerin  agar.     Used  for  the 


186  ATLAS   OF   BACTERIOLOGY. 

culture  of  pathogenic  varieties,  which   thrive   with 
difficulty  or  not  at  all  upon  other  nutrient  media. 
Plate  cultures  are  only  possible  with  glycerin  agar 
and  mixtures  of  agar  and  serum. 
3.  Potatoes. 

(1)  To  obtain  macroscopically  characteristic  cul- 
tures of  great  durability  and  for  differential  diag- 
nosis. 

(2)  Occasionally  for  the  development  of  spores. 

3.  A  Few  Words  on  the  Manipulation  of  Ordinary 
Cultures. 

The  platinum  needle  must  be  brought  to  a  glow 
throughout  its  entire  length  each  time  before  using 
and  before  putting  it  away. 

{a)  Fluid  cultures  are  inoculated  with  a  loopful  of 
pure  culture. 

(b)  Gelatin  and  agar  stick  cultures  are  made  with  a 
straight  needle  without  a  loop,  only  one  puncture  to 
each  tube  but  extending  nearly  to  the  bottom. 

(c)  Agar  and  gelatin  streak  cultures  and  potato  cul- 
tures are  made  by  a  gentle  superficial  stroke  upon  the 
surface  with  the  platinum  loop.  In  the  case  of  the 
potato  it  is  sometimes  necessary  to  rub  the  culture  in. 

(d)  Gelatin  plate  cultures. 

1.  To  isolate  definite  germs  in  the  pure  culture. 
We  melt  three  gelatin  tubes ;  put  into  the  first,  after 
it  has  been  cooled  to  30°,  a  loopful  of  a  fluid  culture 
or  a  trace  of  a  solid  culture;  shake  the  tube  while 
turning  it  upside  down,  and  then  convey  from  this 
one  or  two  loopfuls  of  liquefied  gelatin  into  a  second 
tube.     After  shaking  this,  two  to  three  loopfuls  are 


TECHNICAL  APPENDIX.  187 

placed  in  a  third  tube,  and  tlie  contents  are  then 
poured  into  three  dry  sterilized  plates,  lifting  the 
cover  briefly  and  gently  inclining  the  plate  to  and 
fro,  in  order  that  the  gelatin  may  be  distributed  as 
uniformly  as  possible.  In  making  inoculations  from 
one  tube  to  another  it  is  advisable  to  hold  them  in  an 
inclined  position  in  order  to  guard  against  the  en- 
trance of  foreign  germs.  The  plates  are  then  placed 
in  the  culture  chamber  at  a  constant  temperature  of 
22°  (or  they  may  be  kept  at  the  temperature  of  the 
room)  and  at  the  end  of  two  to  three  days  the  indi- 
vidual colonies  which  have  developed  are  observed 
macroscopically  and  also  microscopically  with  low 
(50)  magnifying  powers.  As  a  general  thing  only 
two  of  the  three  plates  are  serviceable  for  observa- 
tion, one  at  least  is  sown  too  thick  or  too  thin. 

2.  If  we  wish  to  ascertain  the  number  of  colonies, 
for  example,  in  a  specimen  of  water,  we  place  in  three 
test  tubes  of  melted  gelatin,  1  c.c,  0.5  c.c,  and  0.1  c.c. 
of  the  water,  shake  and  pour  into  three  dishes.  To 
ascertain  the  number  of  germs,  we  use  Wolffhiigel's 
counting  apparatus  if  very  many  germs  have  devel- 
oped. If  the  germs  are  few  the  following  plan  is 
simpler:  The  plate  is  laid  upside  down  (upon  the 
cover) ,  the  bottom  is  divided  with  ink  into  sextants, 
and  each  visible  colony  is  marked  with  a  dot.  Plates 
upon  which  the  number  of  germs  in  drinking-water 
are  to  be  ascertained  must  be  counted  several  times 
(on  the  second,  third,  and  fifth  days).  When  the 
fluid  is  very  rich  in  germs  (for  example,  sour  milk, 
ditch  water,  etc.),  1  c.c.  is  first  placed  in  100  c.c.  of 
sterilized  water  and  the  mixture  then  manipulated  as 
described  above.     Solid  bodies  are  first  rubbed  up  in 


188  ATLAS    OF    BACTERIOLOGY. 

water.  "WTien  air  is  to  be  examined  a  definite  volume 
is  sucked  through  a  tube  of  sterilized  sand,  the  latter 
carried  into  sterilized  water,  and  plates  are  then 
formed. 

(e)  Agar  plate  cultures  are  made  in  the  same  way. 
The  agar  should  not  be  poured  into  the  dishes  when 
too  cool,  because  otherwise  it  coagulates  at  once  into 
an  irregular  surface ;  if  used  when  too  warm,  the  in- 
oculated bacteria  will  die*.  In  recent  times  it  has 
been  recommended  that  in  making  agar  plates  the 
nutrient  medium  should  first  be  allowed  to  become 
rigid  in  the  dish,  and  then  the  mass  to  be  examined 
is  smeared  superficially  upon  it  with  a  sterilized 
platinum  loop,  a  strip  of  filtering  paj^er  or  a  xjlatinum 
brush.  In  this  way  we  obtain  only  characteristic 
superficial  colonies. 

(/)  Sugar-agar-agitation  cultures :  The  contents  of 
the  tube  are  melted  in  the  water-bath,  then  cooled  to 
about  40°;  a  loopful  of  pure  culture  is  then  intro- 
duced, the  tube  well  shaken,  and  when  it  becomes 
rigid  the  culture  is  placed  in  the  incubating  chamber. 

4.  Anaerobic  Cultures. 

We  have  employed  almost  exclusively  Buchner's 
method,  i.e.,  the  absorption  of  oxygen  by  pyrogallic 
acid  and  potash  lye.* 

(a)  For  stick  cultures :  Upon  the  bottom  of  a  glass 
cylinder,  which  must  be  somewhat  longer  and  wider 
than  a  test  tube,  is  placed  a  heaping  teaspoonful  of 
pyrogallic  acid  and  20  c.c.  of  a  three-per-cent  potash 

*  Sensitive  varieties  are  said  to  thrive  still  better  in  a  hydrogen 
atmosphere. 


TECHNICAL    APPENDIX.  189 

lye;  place  in  it  the  infected  stick  culture  and  close 
the  cylinder  at  once  with  a  soft-rubber  stopper  or  a 
ground-glass  stopper  which  is  sealed  with  paraffin. 
According  to  Kitasato  the  anaerobics  which  are  less 
sensitive  to  oxygen  may  be  cultivated  in  saccharine 
agar  in  a  high  stick  culture,  even  without  pyrogallic 
acid.  A  wire  with  a  small  loop  is  pushed  into  the 
layer  of  sugar  agar  (8  to  10  c.c.  high),  and  the  wire 
turned  on  its  long  axis  before  withdrawal. 

(b)  For  i^late  cultures  we  use,  instead  of  the  glass 
cylinder,  a  wide  exsiccator  with  a  ground  cover;  fill 
the  lower  part  with  sand  and  the  pyrogallic-acid  mix- 
ture, and  then  manipulate  as  before. 

III.    EXPERIMENTS  ON  ANIMALS. 
A.  Infectioyi. 

1.  Subcutaneous  inoculation.  A  shallow  incision 
is  made  with  a  pair  of  scissors  on  some  part  of  the 
skin,  after  it  has  been  washed  with  a  0.1-per-cent 
solution  of  corrosive  sublimate;  the  inoculating 
matter  is  carried  beneath  the  skin  by  means  of  a 
stout  platinum  wire  with  a  loop.  Mice  are  generally 
inoculated  above  the  root  of  the  tail ;  they  are  simply 
held  by  the  tip  of  the  tail,  and  allowed  to  hang  into  a 
glass  which  is  covered  up  in  great  part  by  a  piece 
of  wood.  Guinea-pigs  and  rabbits  are  inoculated  on 
the  side  of  the  thorax. 

2.  Subcutaneous  injection  is  generally  effected  by 
means  of  Koch's  rubber  ball  injection  syringe  or 
Strohschein's  syringe.  A  fold  of  skin  is  picked  up 
at  some  part  of  the  body,  and  the  needle  inserted  in 
the  longitudinal  direction.     If  several  cubic  centi- 


190  ATLAS   OF   BACTERIOLOGY. 

metres  are  to  be  injected,  the  following  simple  method 
may  be  adopted:  A  short  piece  of  rubber  tube  pro- 
vided with  an  injection  needle  is  fastened  to  a  grad- 
uated pipette,  the  entire  apparatus  sterilized,  the 
pipette  filled,  and  the  fluid  blown'  in  by  the  aid  of  the 
mouth  or  a  rubber  bulb. 

3.  Peritoneal  injection  is  made  by  perforating  with 
a  sterilized  hollow  canula,  at  one  puncture,  the  ab- 
dominal wall,  then  cautiously  advancing  the  needle 
and  injecting  the  fluid. 

B.   Observation. 

Mice  may  be  kept  in  sterilized  glass  vessels  closed 
with  cotton  and  wire  netting ;  larger  animals  must  be 
kept  in  sterilized  cages  or  stalls. 

G.  Autopsy  and  Disposal  of  the  Cadaver. 

Autopsies  must  be  made  immediately  after  death, 
or,  at  least,  the  animal  placed  on  ice.  The  animal, 
lying  on  the  back,  is  tied  or  nailed  through  the  legs 
to  a  board,  the  abdomen  and  chest  are  throughly 
moistened  with  corrosive  sublimate,  and  then  the  ab- 
dominal cavity  is  opened  with  a  previously  sterilized 
knife.  The  abdominal  walls  are  separated  and  from 
the  spleen,  liver,  and  kidneys  some  blood  (or  tissue 
juice)  is  removed  with  a  sterilized  platinum  loop. 
This  is  smeared  at  once  upon  prepared  agar  plates. 
The  organs  are  carefully  cut  out,  avoiding  contact 
with  the  intestines,  and  are  placed  in  absolute  alcohol 
for  further  examination.  Then  the  thorax  is  opened 
with  a  pair  of  scissors,  blood  taken  from  the  heart  and 
lungs,  and  these  organs  are  placed  in  alcohol.  Be- 
fore each  operation  the  instruments  must  be  carefully 


TECHNICAL  APPENDIX.  191 

brought  to  a  glow.  It  is  better  to  have  on  hand 
numerous  instruments  which  have  been  sterilized  at 
130°.     The  hands  must  be  kept  perfectly  clean. 

After  the  autopsy  it  is  best  to  cremate  the  cadaver. 
If  this  is  not  feasible,  the  body  is  wrapped  in  cloths 
dipped  in  a  solution  of  corrosive  sublimate  and  buried 
in  a  hole  in  the  ground  at  least  one-half  metre  deep, 
which  is  filled  in  with  quicklime. 


ALPHABETICAL  INDEX  OF  ILLUSTRATIONS. 


Actinomyces,  pi.  63 
Anthrax  bacillus,  pi.  38-40 
Arthrospores,  pp.  67,  76 
Bacillus  acidi  lactici,  pi.  13 

anthracis,  pi.  38-40 

butyricus,  pi.  42,  V.-VI. 

Chauvoei,  pi.  46 

coli,  pi.  14,  15 

cyanogencs,  pi.  23,  24 

diplitheriae,  pi.  20 

erysipelatos  suum,pl.34,I. 

fluorescens      liquefaciens, 
pi.  28 

fluorescens   non  -  liquefaci- 
ens, pi.  22 

haemorrhagicus,    pi.      21, 
VIL,  VIII. 

influenzae,  pi.  63,  V. 

janthinus,  pi.  27 

kiliensis,  pi.  26 

latericius,  pi.  21,  I. -VI. 

leprae,  pi.  63,  I. -III. 

mallei,  pi.  19 

megatherium,  pi.  35 

mesentericus    fuscus,    pi. 
42,  43,  VIII.,  IX. 

mesentericus  vulgatus,  pi. 
44 

murisepticus,   pi.   34,  II.- 
X. 

13 


Bacillus   mycoldes,  pi.  41-42, 
I.-IV. 

oedematis  maligni,  pi.  47 

pneumonia;,  pi.  12 

prodigiosus,  pi.  25 

putidus,  pi.  22 

pyocyaneus,  pi.  29 

septicacmiae     haemorrhagi- 
cae,  pi.  18 

subtilis.  pi.  36,  37 

syncyaneus,  pi.  24 

tetani,  pi.  45 

typhi,  pi.  16,  17 

violaceus,  pi.  27 

vulgatus,  pi.  43 

Zopfii,  pi.  30,  31 
Bacteria,  forms  of,  p.  66 

in  soft  chancre,  pi.  63,  IV. 
Bacterium  acidi  lactici,  pi.  13 

coli  commune,  pi.  14,  15 

erysipelatos  suum,  pi.  34, 1. 

haemorrhagicum,     pi.    21, 
VIL,  VIII. 

influenzae,  pi.  63,  V. 

janthinum,  pi.  27 

kiliense,  pi.  26 

latericium,  pi.  21,  I. -VI. 

mallei,  pi.  19 

murisepticum,  pi.  34,  II.- 
IX. 


194 


ATLAS   OF   BACTERIOLOGY. 


Bacterium     pediculatum,      p. 
73 
pestis,  pi.  63,  VI.,  VII. 
pneumoniae,  pi.  13 
prodigiosum,  pi.  35 
putidum,  pi.  33 
pyocyaneum,  pi.  39 
septicaemiae     hsemorrbagi- 

cae,  pi.  18 
syncyaneum,  pi.  34 
typhi,  pi.  16,  17 
violaceum,  pi.  37 
vulgare,  pi.  33 
vulgare  /5  mirabilis,  pi.  33 
Zopfii,  pi.  30,  31 
Butyric  acid   bacillus,  pi.  43, 

V.-VII. 
Capsule  bacillus,  Friedlander's, 
pi.  13 
coccus,  Fraenkel's,  pi.  5 
formation  of,  p.  73 
Chain  coccus,  pi.  6 
Chicken  cholera,  pi.  18 
Cholera  bacillus,  pi.  49-53 
reaction,  pi   54,  IV. 
vibrio,  pi.  49-53 
Chromogenous  sarcinae,  pi.  9- 

11 
Cladothrix     dichotoma    Auto- 
rum  non  Cohn,  pi.  61 
Comma  bacillus  of  cholera,  pi. 
49-53 
bacillus  of  Finkler,  pi.  53, 

VI.,  56 
bacillus  of    Metsclmikoff, 
pi.  53,  V. 
Corynebacterium     diphtherise, 

pi.  30 
Diphtheria  bacillus,  pi.  30 


Diplococcus  gonorrhoeae,  pi.  3, 

VL,  Vl.a,  Yl.b 
Diplococcus  lanceolatus,  pi.  5 
pneumoniae,  pi.  5 
roseus,  pi.  4 
Endogenous  spores,  p.  79 
Erysipelas  streptococcus,  pi.  6 
Farcin  de  boeuf,  pi.  60 
Fermentation  tubes,  p.  155 
Finkler 's  comma  bacillus,  pi. 

56,  53,  VI. 
Flagella  types,  p.  73 
Fluorescens  liquefaciens,  pi.  38 

non-liquefaciens,  pi.  33 
Fluorescent    bacteria,    pi.    33, 

38,  39 
Fowl  cholera,  pi.  18 
Fraenkel's  pneumonia  coccus, 

pi.  5 
Friedlander's    pneumonia    ba- 
cillus, pi.  13 
Germination  of  spores,  p.  73 
Glanders  bacillus,  pi.  19 
Gonococcus,  pi.  3,  VI.,  VI. «, 

VL6 
Gonorrhoea,   pi.  3,  VI.,   VI.  a, 

VI.  6 
Green  pus,  pi.  39 
Hanging  drop,  p.  167 
Hauser's  bacterium,  pi.  33,  33 
Hay  bacillus,  pi.  36,  37 
Hog  erysipelas,  pi.  34,  I. 
Indol  reaction  in   cholera,  pi. 

54.  4 
Influenza  bacillus,  pi.  63,  V. 
Involution   forms  of   anthrax, 
pi.  40,  V. 
forms  of  cholera,  pi.  53,  IV. 
Kiel  water  bacillus,  pi.  36 


ALPHABETICAL  INDEX   OF   ILLUSTRATIONS. 


196 


Lactic  acid  bacillus,  pi.  13 
Lepra  bacillus,  pi.  63.  L-IIL 
Leptothrix  epidermidis,  pi.  59 
Loffler's  bacillus,  pi.  20 
Malignant  oedema,  pi.  47 
Malleus,  pi.  19 
Membrane,    thickening   of,  in 

bacteria,  p.  73 
Mesentericus  fuscus,  pi.  44 

vulgatus,  pi.  43 
Metschnikoff's   vibrio,    pi.  53, 

V. 
Micrococcus  agilis,  pi.  3,  I.-V. 
badius,  pi.  11,  VII. 
candicans,  pi.  2,  IV.-VIII. 
gonorrhoeae,  pi.  3,  VI. 
luteus.  pi.  8.  I.-V. 
pyogenes  a  aureus,  pi.  1 
pyogenes  y  albus,   pi.   2, 

I.-IL 
pyogenes  (3  citreus,  pi.  2, 

III. 
roseus,  pi.  4 
tetragenus,  pi.  7 
Morbus  Werlhofii,  pi.  21,  VIL, 

VIII. 
Mouse  septicaemia,  pi.  34 

typhoid,  pi.  17,  XL 
Mycobacterium  leprae,  pi.  63, 
L-IIL 
tuberculosis,  pi.  48 
Oospora  bo  vis,  pi.  62 
chromogenes,  pi.  61 
farcinlca,  pi.  60 
Pediococcus  tetragenus,  pi.  7 
Plague   bacillus,   pi.   63,   VL, 

VIL 
Plasmolysis,       according       to 
Fischer,  70 


Pneumonia  bacillus,  pi.  12 

coccus,  pi.  5 
Potato  bacillus,  pi.  42,  VIII. , 

IX.,  43,  44 
Prodigiosus,  pi.  25 
Proteus  mirabilis,  pi.  32 

vulgaris,  pi.  33 
Pseudodichotomy    in    bacilli, 
69 
in  streptococci,  69 
Pus,  green,  blue,  pi.  29 
Pyocyaneus,  pi.  29 
Rabbit  septicaemia,  pi.  18 
Rauschbrand,  pi,  46 
Recurrens     spirilli,     pi.      58, 

VIIL,  IX. 
Root  bacillus,  pi. 41,  42,  L-IV. 
Sarcina  aurantiaca,  pi.  10. 
canescens,  pi.  11,  VIIL 
cervina,  pi.  11,  I. 
erythromyxa,  pi.  11,  III. 
flava,  pi.  9 
lutea,  pi.  11,  IV. 
pulmonum,  pi.  8 
rosea,  pi.  11,  VL 
Septicaemia  haemorrhagica,  pi. 

18 
Spirilli  from  the  gums,  pi.  58, 
VII. 
from    the    nasal    mucous 
membrane,  pi.  58,  III., 
IV. 
Spirillum    concentricum,    pi. 

57,  VI. ,  VIIL 
Spirillum  Obermeieri,  pi.  58, 
VIIL,  IX. 
rubrum,  pi.  47,  I.-V. a 
serpens,  pi.  58,  I. 
undula,  pi.  58,  V. 


196 


ATLAS   OF   BACTERIOLOGY. 


Spirocbtete  Obermeieri,  pi,  58, 
VIII.,  IX. 
of  the  gums,  pi.  58,  VII. 
Spores,  development  of,  77 
germiDation  of,  78 
types  of,  77 
Staphylococcus    pyogenes    al- 
bus,  pi.  2,  L,IL 
pyogenes  aureus,  pi.  1. 
pyogenes    citreus,    pi.    2, 
III. 
Streptococcus  brevis,  pi.  6,  X. 
conglomerates,  pi.  6,  XI. 
longus,  pi.  6,  IX. 
meningitidis      cerebrospi- 
nal is,  pi.  3,  VIL,  VIII. 
of  erysipelas,  pi.  6 
Streptococcus  pyogenes,  pi.  6 
Streptothrix,  pi.  60 


Structure    of    the    bacterium 
cell,  70 

Tetanus  bacillus,  pi.  45. 

Tetragenus,  pi.  7 

Tuberculosis,  pi.  48 

Typhoid  bacillus,  pi.  16,  17 

Vibrio  albensis,  pi.  54 

aquatilis,  pi.  55,  II.,  VIL, 

VIII.,  IX. 
berolinensis,pl.  55,  V.,  VI. 
cholerse,  pi.  49-53 
danubicus,  pi.  55,  I. -III. 
Finkler,  pi.  53,  VI.,  56 
fluorescent,  from  the  Elbe, 

pi.  54 
Metschnikoff,  pi.  53,  V. 
proteus,  pi.  53,  VI.,  56 
spermatozoides,  pi.  58,  VI. 

Violet  bacillus,  pi.  37 


IWDEX 


Abbe's    illuminating    appara- 
tus, 166 
Abrin,  135,  163 
Absolute  immunity,  157 
Acclimatization  of  anthrax,  99 
Aceton,  150 
Acid,  acetic,  150 

agar,  89 

butyric,  150 

formic,  150 

media,  use  of,  90 

propionic,  150 
Active  immunization,  157 
Adenin,  81 

Al^robic  races  of  anaerobic  va- 
rieties, 97 
Aerobics,  facultative,  96 

strict,  95 
Aerotaxic  figures,  112 
^thyl  alcohol,  149 
Agar*  cultures,  189 
Albuminoids    in    bacteria,   80 

labile,  135 
Alcohol,  150 

production  of  acids  from, 
156 
Aldehyde,  150 
Agitation  cultures,  101 
Alexin,  163 

Alkali,  production  of,  by  bac- 
teria, 130 


Alkaline  agar,  89 
Alkaloids,  putrefaction,  132 
Alternating  fission  in  different 

planes,  75 
Alum  carmine,  169 
Amidoacids,  133 
Amines.  130,  133 
Ammonia,    demonstration    of, 
141 

production  of,  130,  141 
Ammonium  bases,  133 

carbonate    in   water   as  a 
nutrient,  85 
Amygdalin,  123 
Anaerobic  cultures,  188 
Anaerobics,  conversion  of,  into 
aerobics,  97 

facultative,  96 

strict,  96 
Aniline  fuchsin,  168 

gentian,  169 

oil,  169 

water,  169 
Animals,  experiments  on,  189 
Antagonistic  action  in  the  ani- 
mal body,  157 

bacteria,  104 
Anthrax  spores,  viability  of, 

108 
Antisepsis,  90 
Antisubstances,  164 


198 


INDEX. 


Antitoxic  effects,  164 

Antitoxin,  164 

Aromatic    metabolic   products 

of  bacteria,  142 
Arthrospores,  67.  76 
Ascitic  fluid,  159 
Asepsis,  90 
Ash,   amount  of,  in  bacteria, 

80 
Assimilation  of  nitrogen,  147 
Attenuation  of  spores,  159, 
of  virulence,  90,  159 

Bacillus  ^thaceticus,  157 
amylobar^ter,  153 
anthracis,  79,  97,  99,  102, 

122,  141,  145,  159 
aquatilis,  85 

butyrious  Hilppe,  81,  152 
Cliauvoei,  96 
De  Baryanus,  77 
denitrificans  I.,  147 
denitrificans  II,,  147 
diplitheriae,  157 
crythrosporus,  85 
fluorescens      liquefaciens, 

122,  132,  140 
kiliense.  122 
leptosporus,  79 
limosus,  77 
macrosporus,  77 
megatherium,  111,  122 
mesentericus,  99,  113 
raycoides,  101,  145 
oedematis    maligni,    96, 

161 
oxalaticus,  71 
perlibratus.  113 
radicicola,  147 


Bacillus  sessilis,  80 

Solmsii,  77 

subtilis,  80,  101,  111,  113, 
120,  141 

tetani,  87,  96,  106 

thermophilus,  99 

tuberculosis, 

urese,  131 

viscosus  sacchari,  81 

vulgatus,  98 
Bacteria,  antagonism  between, 
104 

chemiccl   composition  of, 
80 

chemical  effects,  115 

definition,  65 

growth  in  groups,  67 

mechanical   and  electrical 
effects  of,  100 

mechanical  effects,  111 

optical  effects,  111 

resistance  of,  to  deficiency 
of  food  and  water,  93 

solitary  growth  of,  67 

thermic  effects,  115 

vital  conditions  of,  84 
Bacterial  proteins,  135 
Bacterio-fluorescin,  128 
Bacterio-trypsin,  117      , 
Bacteroids,  148 
Bacterium  aceti,  156 

acidi  lactici,  86,  97 

Bischleri,  152 

cholerse  gallinarum,  94 

coli,  110,  141,  145,  147 

cuniculicida,  87 

erysipelatos  suum,  87 

indigonaceum,  128 

janthinum,  128 


IKDEX. 


199 


Bacterium  kiliense,  127,  130 
mallei,  122 
murisepticum,  87 
pediculatum,  73 
PflUgeri,  98,  105 
phosphorescens,  114 
pneumonice,  82,  122 
prodigiosum,  82,  102,  121, 

145 
putidum,  102,  104 
pyocyaneum,  121 
pyogenes  fatidum,  122 
syncyaneum,  129 
synxanthium,  122 
typhi,  145,  147 
violaceum,  122,  127 
vulgare,  119,  160 
vulgare  /9  Zenkeri,  144 

Beer  wort,  180 

Beggiatoa,  81 

Beozaldehyde,  123 

Bilineurin,  133 

Bismarck  brown,  169 

Blood  serum,  183 

Blue  milk,  128 

Bouillon  culture,  141 

Brieger's  method  of  isolating 
ptomains,  134 

Brownian     molecular     move- 
ments, 112 

Bunge's  granules,  71 
mordant,  170 

Butter,  rancidity  of,  143 

Butyl  alcohol,  152 

Butyric  acid,  152 

Cadaverin,  133 
Capsule  bacteria,  72 
preparation  of,  172 


Carbohydrates,   production  of 

acids  from,  148 
Carbolized  fuchsin,  168 
Carbonic  acid,  action  on  bac- 
teria, 97 
Carolin,  127 
Cedar,  oil  of,  167 
Cell  structure  of  bacteria,  68 
Cellulose,  81 

decomposition  of,  by  bac- 
teria, 153 
Central  body  of  bacteria,  71 

fluid  of  bacteria,  70 
Chemical  composition  of  bac- 
teria, 80 

effects  of  bacteria,  115 

ferments,  116 
Chemotaxis,  112 
Cholera  as  a  nitrite  poisoning, 
158 

diblastic  theory  of,  106 
Cholesterin,  80 
Cholinbilineurin,  133 
Chromogenic  functions  of  bac- 
teria, 129 
Chromogenous  bacteria,  110 
Cinnamic  acid,  163 
Clostridium  butyricum,  152 
Club-shaped  bacteria,  67 
Comma  bacteria,  67 
Congenital  immunity,  162 
Counting  bacteria,  105 
Creolin,  92 
Cultures,  179 

manipulation  of,  186 

anaerobic,  188 

Decomposition  of  cellulose  by 
bacteria,  153 


200 


INDEX. 


Decomposition  of  fats,  143 

Definition  of  bacteria,  65 

Degeneration    forms    of    bac- 
teria, 80 

Demonstration  of  indol,  142 
of  nitrites,  141 
of  phenol,  143 

Desiccation  experiments,  94 

Deuteroalbumose,  135 

Diastatic  ferments,  121 

Dichotomy,  68 

Diethylamin,  133 

Dimethylamin,  133 

Dimethylethylendiamin,  133 

Diphtheria  antitoxin,  164 

Disinfectants,  combination  of, 
90,  93 

Distilled  water,  action  on  bac- 
teria, 93 

Dry     bacteria,     viability     of, 
94 

Drying  nutrient  media,  93 

Dulcite,  156 

Ehrlicii's  solution,  169 
Electric    arc    action   on    bac- 
teria, 102 
Enantobiosis,  104 
Endospores,  76 

staining  of,  174 
Enzymes,  116 

proteolytic,  117 
Ernst's  granules,  71 
Ethyl,  150 
Ethylamin,  133 
Ethylendiamin,  133 
Ethylidlactic  acid,  151 
Eubacillus  multisporus,  66 
Experiments  on  animals,  189 


Extractive  matters  in  bacteria, 
80 

Facultative  aerobics,  96 

anaerobics,  96 
Fats,  decomposition  of,  143 
Fermentation,     definition     of, 
124 

flask,  155 

lactic  acid,  151 

oxidative,  125 
Ferments,  116 

diastatic,  121 

inverting,  122 

rennet,  123 
Ferric  oxide,  81 
Fibrin,  liquefaction  of,  117 
Filamentous  bacteria,  67 
Fission  of  bacteria,  75 
Flagella,  73 

mordants,  169 

staining  of.  172 
Flagellates,  65 
Flesh-water    peptone   gelatin, 

87 
Fluorescent  pigments,  126 
Formic  acid,  156 
Frog -spawn  disease,  73 
Fuchsin,  168 

Gas,  formation  of,  from  carbo- 
hydrates, 153 
Gelatin,  liquefaction   of,  117, 
119 
neutral,  88 
nutrient  media,  181 
various  kinds  of,  181,  182 
Germination  of  spores,  78 
Globulin  in  bacteria,  80 


INDEX. 


201 


Gly cerate  of  lime,  157 
Glycerin,  156 

agar,  87,  183 
Gram's  stain,  171 
Granules,  Bunge's,  71 

Ernst's,  71 

metachromatic,  71 

sporogenous,  71 
Granulobacter  polymyxa  Bey- 

erinck,  152 
Growth  of  bacteria,  67 
Guanidin,  133 
Guanin,  81 

HALF-scREW-shaped  bacteria, 
67 

Hanging  drop,  167 

Hay  decoction,  180 

Heat,  production  by  bacteria, 
115 

Hemicellulose,  81 

Herring  gelatin,  182 

Honeycomb  structure  of  bac- 
teria, 69 

Hydrocarbons  in  bacteria,  81 

Hydrogen  peroxide,  produc- 
tion on  illuminated  cultures, 
103 

Immune  proteidins,  164 
Immunity,  157 
Increase  of  virulence,  160 
Indicator,  88 
Indol,  133 

demonstration  of,  142 
Inverting  ferments,  122 
Involution  forms  of  bacteria,  80 
Iodine -potassium    iodide  solu- 
tion, 169 


Iodoform,  150 
Iris  diaphragm,  166 
Isatin  sulphate,  140 
Isolation  of  ptomains,  134 

Knob  bacteria,  147 
Koch's  tuberculin,  135 
Koly sepsis,  90 

Labile  albuminoids,  135 
Lactate  of  lime,  157 
Lactic-acid  fermentation,  151 
Lecithin,  80 
Leptothrix,  81 
Leucin,  133 

Leuconostoc  mesenterioides,  81 
Leuko  substances,  140 
Lieber's    iodoform     reaction, 

150 
Lime,  glycerate  of,  157 

lactate  of,  157 
Lipochromata,  127 
Liquefaction  of  gelatin,  119 
Litmus,  reduction  of,  140 

whey,  180 
LOffler's  methyl  blue,  169 

mordant,  169 
Longitudinal  fission,  75 
Long  rod-shaped  bacteria,  67 

screw-shaped  bacteria,  67 

Malignant  oedema,  viability 

of  spores,  108 
Mallein,  135 
Mannite,  156 
Marsh  gas,  145,  153 
Membrane  of  bacteria,  68 
Mercaptan,  139 
Mesophilic  bacteria,  99 


202 


INDEX. 


Metachromatic  granules,  71 
Metaphenylendiamin,  141 
Methylamin,  133 
Methyl  blue,  168 

guanidin  poisoning,  158 
Micrococcus   acidi  paralactici, 
153 

agilis,  112 

cereus  flavus,  126 

gonorrhoeae,  85 

mastitidis,  122 

pyogenes,  104,  119,  157 

tenuis,  123 

tetragenus,  122,  161 

urese  Leube,  131 
Microscopical  technique,  166 
Milk,  180 

ferment,  116 
Mitigation  of  virulence,  90 
Mordant,  Bunge's,  170 

Loffler's,  169 
Motile    bacteria,     sporulation 

of,  77 
Motion   of  bacteria,   character 

of.  111 
Muscarin,  133 

Naphthylamin,  141 
Negative  chemotaxis,  112 
Neuridin,  133 
Neutral  agar,  89 

bacteria,  148 

gelatin,  88 
Nicolle's  stain,  177 
Nitrates,  reduction  of,  140 
Nitric    acid,    conversion    into 

free  acid,  147 
Nitrification,  145 
Nitrite  poisoning,  158 


Nitrites,  demonstration  of,  141 
Nitrogen,  assimilation  of,  147 
Nitrosobacter,  146 
Nitrosomonas,  146 
Non-albuminous  nutrient  me- 
dia, 179 
Normal  soda,  88 
Nuclein,  81 
Nucleus  of  bacteria,  69 
Nutrient  agar,  182 

bouillon,  180 
Nutrient  media,  84,  179 

acid,  89 

albuminous,  117,  180 

alkaline,  87 

employment  of,  184 

gelatin,  181 

neutral,  87 

non-albuminous,  121,  179 

saccharine,  122 

Oil  immersion  lens,  166 

of  cedar,  167 
Optical  effects  of  bacteria.  111 
Oval  bacteria,  67 
Oxidative  fermentation,  125 
Oxyfatty  acids,  144 

Papayotin,  163 
Parvolin,  133 
Pasteuria,  75 
Pathogenic  bacteria,  110 
Pathogenesis,  157 
Pentamethylendiamin,  133 
Peptone  water,  180 
Peptones,  118 
Phagocytosis,  163 
Phenolphthalein,  88 
Phlogogenic  albuminoids,  135 


INDEX. 


203 


Phosphorescent  bacteria,  113 
Photobacteriura,  114 
Phycochromacea,  65 
Pigment,  formation  of,  126 
Plasma  of  bacteria,  69 
Plasmolysis,  70 
Polar  flagella,  73 
Positive  chemotaxis,  112 

thermotropism,  113 
Predisposition,  157 
Processes  of  reduction,  140 
Production  of  acids  from  alco- 
hols, 156 

of    acids    from    carbohy- 
drates, 148 
Proteidins,  immune,  164 
Proteolytic  ferments,  117 
Pseudodichotomy,  68 
Pseudopodia,  73 
Psychrophilic  bacteria,  99 
Ptomains,  133 
Putrefaction,  144 

alkaloids,  133 
Putrescin,  133 
Pyogenic  albuminoids,  135 
Pyridin,  133 

Rabbit  septicaemia,  158 
Rancidity  of  butter,  143 
Ranges  of  temperature  for  bac- 
teria, 98 
Reaction    of    nutrient    media, 

87 
Red  pigments,  136 
Reduction  of  nitrates,  140 

processes,  140 
Relative  immunity,  161 
Rennet  ferments,  133 
Resistance,  157 


Resistance  of  bacteria  to   de- 
ficiency of  food  and  wat- 
er, 93 
of  spores,  108 

Ricin  135 

Rinderpest,  161 

Saline  solutions  as  nutrients, 

86 
Saprogenous  bacteria,  110 
Saprophytes,  85 
Sarcina  pulmonum,  106 
Schizomycetes,  65 
Section  preparations,  176 
Separation  of  acids  produced 

by  bacteria,  150 
Sepsm,  133 
Short  rod -shaped  bacteria,  67 

screw -shaped  bacteria,  67 
Silicic  acid  nutrient  medium, 

184 
Simple  nutrient  media,  85 
Skatol,  133 

Smear  preparations,  170 
Solitary  growth  of  bacteria,  67 
Spermin,  163 
Spherical  bacteria,  67 
Spindle-shaped  bacteria,  67 
Spirillum  desulphuricans,  139 

endoparagocicum,  107 
Spores,  attenuation  of,  159 

biological    characters    of, 
106 

germination  of,  78 

power  of  resistance  of,  107 

tests  for,  109 
Sporogenous  granules,  71 
Sporulation,  77 

influences  favoring,  107 


204 


INDEX. 


Sporule,  preliminary  stage,   71 
Staining  solutions,  168 
Stellate  fission,  75 
Sterilization,  90 
Strict  ae^robics,  95 

anaerobics,  96 
Succinic  acid,  156 
Sugar,  chalk  agar,  183 

fermentation  of,  125 
Sulphanilic  acid,  141 
Sulphates,  139 
Sulphmethsemoglobin,  158 
Sulphur  granules,  81 
Sulphuretted     hydrogen,    98, 

138 
Sunlight,    action   on   bacteria, 

101 
Susceptibility,  161 
Symbiosis,  104 
Syncyanin,  128 
Synergetic  bacteria,  104 

Tests  of  disinfectants,  91 
Tetanus  antitoxin,  164 

poison,  136 

spores,  viability  of,  108 

virulence  of,  137 
Tetrads,  68 

Thermophilic  bacteria,  99 
Thermotropism,  113 
Thiosulphite,  139 
Titration,  88 
Torula,  68 

Toxalbumins,  134,  136 
Toxins,  132,  134  j 

Transverse  fission  of  bacteria,   | 
75  I 


Trimethylamin,  133 

Triolein,  80 

Tripalmitin,  80 

Tristearin,  80 

Tubercle  bacilli,   staining  of, 

175 
Tuberculin,  Koch's,  135 
Tyrosin,  133 
Tyrothrix  tenuis,  171 

Universal  nutrient,  89 
Urea  fermentation,  131 
Uschinsky  solution,  86 

Vegetative  proliferation,  66, 

75 
Viability  of  dry  bacteria,  94 

of  spores,  108 
Vinylcholin,  133 
Violet  pigments,  127 
Virulence  oi  bacteria,  attenua- 
tion of,  159 
increase  of,  160 
Vital    conditions  of  bacteria, 
84 

Water  bacteria,  85 

Xanthin,  81 
Xylol,  168 

Yellow  pigments,  126 

Ziehl's  solution,  168 
ZoSglcea.  73 
Zymogenous  spores,  110 


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